A decrease in dkk1, a Wnt inhibitor, contributes to placental and fetal lipid accumulation in an obesity-prone rat model

Placenta, as the sole transport mechanism between mother and fetus, links the maternal physical state and the immediate and life-long outcomes of the offspring. The present study examined the mechanisms behind the effect of maternal obesity on placental lipid accumulation and metabolism. Pregnant Obese Prone (OP) and Obese Resistant (OR) rat strains were fed a control diet throughout gestation. Placentas were collected on gestational d21 for analysis and frozen placental sections were analyzed for fat accumulation as well as β-Catenin and Dkk1 localization. Additionally, DKK1 was overexpressed in JEG3 trophoblast cells, followed by treatment with NEFA and Oil Red O stain quantification and mRNA analysis to determine the relationship between placental DKK1 and lipid accumulation. Maternal plasma and placental NEFA and TG were elevated in OP dams, and offspring of OP dams were smaller than OR. Placental Dkk1 mRNA content was 4-fold lower in OP placentas, and there was a significant increase in β-Catenin accumulation as well as mRNA content of fat transport and TG synthesis enzymes, including Ppar-delta, Fatp1, Fat/Cd36, Lipin1, and Lipin3. There was significant lipid accumulation within the decidual zones in OP but not OR placentas, and the thickness of the decidual and junctional zones was significantly smaller in OP than OR placentas. Overexpression of DKK1 in JEG3 cells decreased lipid accumulation and the mRNA content of PPAR-Delta, FATP1, FAT/CD36, LIPIN1, and LIPIN3. Our results indicate that Dkk1 may be regulating placental lipid metabolism through Wnt-mediated mechanisms. Additionally, recent studies have suggested that maternal obesity may also program early development of non-alcoholic fatty liver disease (NAFLD), rates of which have correlated with the increase in the obesity epidemic. In the current study, livers of OP offspring had significantly increased TG content (P<0.05) and lipid accumulation when compared to offspring of OR dams. Additionally, hepatic Dkk1 mRNA content was significantly decreased in OP livers when compared to OR (P<0.05), and treating H4IIECR rat hepatocyte cells with NEFA showed that Dkk1 mRNA was also decreased in NEFA-treated cells (P<0.05) that also had lipid accumulation. Chromatin Immunoprecipitation (ChIP) analysis of the Dkk1 promoter in fetal livers showed a pattern of histone modifications associated with decreased gene transcription in OP offspring, which agrees with our gene expression data. These results demonstrate that the hepatic Dkk1 gene is epigenetically regulated via histone modification in neonatal offspring in the current model of gestational obesity, and future studies will be needed to determine whether these changes contribute to excessive hepatic lipid accumulation in offspring of obese dams.

Dual regulation of P-glycoprotein expression by Trichostatin A in cancer cell lines

Background. It has been reported that the histone deacetylase inhibitor (iHDAc) trichostatin A (TSA) induces an increase in MDR1 gene transcription (ABCB1). This result would compromise the use of iHDACs in combination with other cytotoxic agents that are substrates of P-glycoprotein (Pgp). It has also been reported the use of alternative promoters by the ABCB1 gene and the existence of a traslational control of Pgp protein. Finally, the ABCB1 gene is located in a genetic locus with the nested gene RUNDC3B in the complementary DNA strand, raising the possibility that RUNDC3B expression could interfere with ABCB1 alternative promoter regulation. Methods. A combination of RT-PCR, real time RT-PCR, Western blot and drug accumulation assays by flow cytometry have been used in this study. Results. The iHDACs-induced increase in MDR1 mRNA levels is not followed by a subsequent increase in Pgp protein levels or activity in several pancreatic and colon carcinoma cell lines, suggesting a traslational control of Pgp in these cell lines. In addition, the MDR1 mRNA produced in these cell lines is shorter in its 5' end that the Pgp mRNA produced in cell lines expressing Pgp protein. The different size of the Pgp mRNA is due to the use of alternative promoters. We also demonstrate that these promoters are differentially regulated by TSA. The translational blockade of Pgp mRNA in the pancreatic carcinoma cell lines could be related to alterations in the 5' end of the MDR1 mRNA in the Pgp protein expressing cell lines. In addition, we demonstrate that the ABCB1 nested gene RUNDC3B expression although upregulated by TSA is independent of the ABCB1 alternative promoter used. Conclusions. The results show that the increase in MDR1 mRNA expression after iHDACs treatment is clinically irrelevant since this mRNA does not render an active Pgp protein, at least in colon and pancreatic cancer cell lines. Furthermore, we have demonstrated that TSA in fact, differentially regulates both ABCB1 promoters, downregulating the upstream promoter that is responsible for active P-glycoprotein expression. These results suggest that iHDACs such as TSA may in fact potentiate the effects of antitumoral drugs that are substrates of Pgp. Finally, we have also demonstrate that TSA upregulates RUNDC3B mRNA independently of the ABCB1 promoter in use.

RGS14 (414)-mediated prevention of an episodic memory loss: a study of molecular mechanism

A large proportion of human populations suffer memory impairments either caused by normal aging or afflicted by diverse neurological and neurodegenerative diseases. Memory enhancers and other drugs tested so far against memory loss have failed to produce therapeutic efficacy in clinical trials and thus, there is a need to find remedy for this mental disorder. In search for cure of memory loss, our laboratory discovered a robust memory enhancer called RGS14(414). A treatment in brain with its gene produces an enduring effect on memory that lasts for lifetime of rats. Therefore, current thesis work was designed to investigate whether RGS14(414) treatment can prevent memory loss and furthermore, explore through biological processes responsible for RGS-mediated memory enhancement. We found that RGS14(414) gene treatment prevented episodic memory loss in rodent models of normal aging and Alzheimer´s disease. A memory loss was observed in normal rats at 18 months of age; however, when they were treated with RGS14(414) gene at 3 months of age, they abrogated this deficit and their memory remained intact till the age of 22 months. In addition to normal aging rats, effect of memory enhancer treatment in mice model of Alzheimer´s disease (AD-mice) produced a similar effect. AD-mice subjected to treatment with RGS14(414) gene at the age of 2 months, a period when memory was intact, showed not only a prevention in memory loss observed at 4 months of age but also they were able to maintain normal memory after 6 months of the treatment. We posit that long-lasting effect on memory enhancement and prevention of memory loss mediated through RGS14(414) might be due to a permanent structural change caused by a surge in neuronal connections and enhanced neuronal remodeling, key processes for long-term memory formation. A neuronal arborization analysis of both pyramidal and non-pyramidal neurons in brain of RGS14(414)-treated rats exhibited robust rise in neurites outgrowth of both kind of cells, and an increment in number of branching from the apical dendrite of pyramidal neurons, reaching to almost three times of the control animals. To further understand of underlying mechanism by which RGS14(414) induces neuronal arborization, we investigated into neurotrophic factors. We observed that RGS14 treatment induces a selective increase in BDNF. Role of BDNF in neuronal arborization, as well as its implication in learning and memory processes is well described. In addition, our results showing a dynamic expression pattern of BDNF during ORM processing that overlapped with memory consolidation further support the idea of the implication of this neurotrophin in formation of long-term memory in RGS-animals. On the other hand, in studies of expression profiling of RGS-treated animals, we have demonstrated that 14-3-3ζ protein displays a coherent relationship to RGS-mediated ORM enhancement. Recent studies have demonstrated that the interaction of receptor for activated protein kinase 1 (RACK1) with 14-3-3ζ is essential for its nuclear translocation, where RACK1-14-3-3ζ complex binds at promotor IV region of BDNF and promotes an increase in BDNF gene transcription. These observations suggest that 14-3-3ζ might regulate the elevated level of BDNF seen in RGS14(414) gene treated animals. Therefore, it seems that RGS-mediated surge in 14-3-3ζ causes elevated BDNF synthesis needed for neuronal arborization and enhanced ORM. The prevention of memory loss might be mediated through a restoration in BDNF and 14-3-3ζ protein levels, which are significantly decreased in aging and Alzheimer’s disease. Additionally, our results demonstrate that RGS14(414) treatment could be a viable strategy against episodic memory loss.

Activité physique et récompense : impact de la leptine et de la signalisation STAT3 dans les neurones dopaminergiques

La course d’endurance active le système de récompense (SR) et est reliée aux comportements de recherche alimentaire. L’influence de la leptine sur l’activité physique (AP) volontaire est bien documentée d’un point de vue physiologique, mais très peu en termes d’impact hédonique. La leptine inhibe l’effet récompensant lié à la consommation de nourriture et joue un rôle semblable pour d’autres types de stimuli. La leptine s’arrime à la forme longue du récepteur à la leptine (Leprb) situé sur les neurones à dopamine (DA) et GABA de l’aire tegmentale ventrale (ATV) dans le mésencéphale. Signal transducer and Activator of Transcription 3 (STAT3) est un facteur de transcription important de la cascade de signalisation de la leptine. La phosphorylation de STAT3 n’est détectée que dans une parcelle des neurones DA positifs pour le Leprb, conférant aux neurones DA STAT3-spécifiques des caractéristiques uniques. Nous avons généré un modèle murin invalidé pour STAT3 sélectivement dans les neurones DA (STAT3DAT-KO). La première expérience consistait à évaluer les paramètres métaboliques de base de notre modèle en utilisant les chambres métaboliques Comprehensive Lab Animal Monitoring System (CLAMS), incluant l’activité ambulatoire, le ratio d’échanges respiratoires (RER) et la production de chaleur. Les STAT3DAT-KO sont hyperactives, démontré par une activité locomotrice augmentée, mais aucune variation entre les deux groupes n’est observée pour le RER et la production de chaleur, en plus d’un gain de poids identique. Une stratégie de récupération ciblant la réinsertion de STAT3 dans les neurones DA du système mésolimbique normalise l’AP anciennement plus élevée des STAT3DAT-KO à celle des contrôles, suivant l’accès libre à une roue d’exercice (RE) pour une durée de 4 semaines, suivant l’accès libre à une roue d’exercice (RE) pour une durée de 4 semaines. L’injection d’un psychostimulant (agoniste du récepteur DA de type 1 (D1R), le Chloro-APB-Hydrobromide (SKF 82958)) reflète une fonction dopaminergique réduite chez les STAT3DAT-KO. Un test de recherche compulsive de nourriture révèle une suppression de la prise alimentaire chez les deux groupes expérimentaux. Nous démontrons pour la première fois que la motivation alliée à la course d’endurance, indépendamment de la régulation de la prise alimentaire par la leptine, est dépendant d’une signalisation leptine-STAT3 amoindrie dans les neurones DA du système mésolimbique, révélant STAT3 comme élément clé dans la régulation du tonus dopaminergique et des propriétés récompensantes de l’AP.

Understanding ten-eleven translocation-2 in hematological and nervous systems

I proposed the study of two distinct aspects of Ten-Eleven Translocation 2 (TET2) protein for understanding specific functions in different body systems. ^ In Part I, I characterized the molecular mechanisms of Tet2 in the hematological system. As the second member of Ten-Eleven Translocation protein family, TET2 is frequently mutated in leukemic patients. Previous studies have shown that the TET2 mutations frequently occur in 20% myelodysplastic syndrome/myeloproliferative neoplasm (MDS/MPN), 10% T-cell lymphoma leukemia and 2% B-cell lymphoma leukemia. Genetic mouse models also display distinct phenotypes of various types of hematological malignancies. I performed 5-hydroxymethylcytosine (5hmC) chromatin immunoprecipitation sequencing (ChIP-Seq) and RNA sequencing (RNA-Seq) of hematopoietic stem/progenitor cells to determine whether the deletion of Tet2 can affect the abundance of 5hmC at myeloid, T-cell and B-cell specific gene transcription start sites, which ultimately result in various hematological malignancies. Subsequent Exome sequencing (Exome-Seq) showed that disease-specific genes are mutated in different types of tumors, which suggests that TET2 may protect the genome from being mutated. The direct interaction between TET2 and Mutator S Homolog 6 (MSH6) protein suggests TET2 is involved in DNA mismatch repair. Finally, in vivo mismatch repair studies show that the loss of Tet2 causes a mutator phenotype. Taken together, my data indicate that TET2 binds to MSH6 to protect genome integrity. ^ In Part II, I intended to better understand the role of Tet2 in the nervous system. 5-hydroxymethylcytosine regulates epigenetic modification during neurodevelopment and aging. Thus, Tet2 may play a critical role in regulating adult neurogenesis. To examine the physiological significance of Tet2 in the nervous system, I first showed that the deletion of Tet2 reduces the 5hmC levels in neural stem cells. Mice lacking Tet2 show abnormal hippocampal neurogenesis along with 5hmC alternations at different gene promoters and corresponding gene expression downregulation. Through the luciferase reporter assay, two neural factors Neurogenic differentiation 1 (NeuroD1) and Glial fibrillary acidic protein (Gfap) were down-regulated in Tet2 knockout cells. My results suggest that Tet2 regulates neural stem/progenitor cell proliferation and differentiation in adult brain.^

Activité physique et récompense : impact de la leptine et de la signalisation STAT3 dans les neurones dopaminergiques

La course d’endurance active le système de récompense (SR) et est reliée aux comportements de recherche alimentaire. L’influence de la leptine sur l’activité physique (AP) volontaire est bien documentée d’un point de vue physiologique, mais très peu en termes d’impact hédonique. La leptine inhibe l’effet récompensant lié à la consommation de nourriture et joue un rôle semblable pour d’autres types de stimuli. La leptine s’arrime à la forme longue du récepteur à la leptine (Leprb) situé sur les neurones à dopamine (DA) et GABA de l’aire tegmentale ventrale (ATV) dans le mésencéphale. Signal transducer and Activator of Transcription 3 (STAT3) est un facteur de transcription important de la cascade de signalisation de la leptine. La phosphorylation de STAT3 n’est détectée que dans une parcelle des neurones DA positifs pour le Leprb, conférant aux neurones DA STAT3-spécifiques des caractéristiques uniques. Nous avons généré un modèle murin invalidé pour STAT3 sélectivement dans les neurones DA (STAT3DAT-KO). La première expérience consistait à évaluer les paramètres métaboliques de base de notre modèle en utilisant les chambres métaboliques Comprehensive Lab Animal Monitoring System (CLAMS), incluant l’activité ambulatoire, le ratio d’échanges respiratoires (RER) et la production de chaleur. Les STAT3DAT-KO sont hyperactives, démontré par une activité locomotrice augmentée, mais aucune variation entre les deux groupes n’est observée pour le RER et la production de chaleur, en plus d’un gain de poids identique. Une stratégie de récupération ciblant la réinsertion de STAT3 dans les neurones DA du système mésolimbique normalise l’AP anciennement plus élevée des STAT3DAT-KO à celle des contrôles, suivant l’accès libre à une roue d’exercice (RE) pour une durée de 4 semaines, suivant l’accès libre à une roue d’exercice (RE) pour une durée de 4 semaines. L’injection d’un psychostimulant (agoniste du récepteur DA de type 1 (D1R), le Chloro-APB-Hydrobromide (SKF 82958)) reflète une fonction dopaminergique réduite chez les STAT3DAT-KO. Un test de recherche compulsive de nourriture révèle une suppression de la prise alimentaire chez les deux groupes expérimentaux. Nous démontrons pour la première fois que la motivation alliée à la course d’endurance, indépendamment de la régulation de la prise alimentaire par la leptine, est dépendant d’une signalisation leptine-STAT3 amoindrie dans les neurones DA du système mésolimbique, révélant STAT3 comme élément clé dans la régulation du tonus dopaminergique et des propriétés récompensantes de l’AP.

An investigation of the relationship between the components of tumour microenvironment, signal transduction pathways and outcome in breast cancer

Breast cancer, the most commonly diagnosed type of cancer in women, is a major cause of morbidity and mortality in the western world. Well-established risk factors of breast cancer are mostly related to women’s reproductive history, such as early menarche, late first pregnancy and late menopause. Survival rates have improved due to a combination of factors, including better health education, early detection with large-scale use of screening mammogram, improved surgical techniques, as well as widespread use of adjuvant therapy. At initial presentation, clinicopathological features of breast cancer such as age, nodal status, tumour size, tumour grade, and hormonal receptor status are considered to be the standard prognostic and predictive markers of patient survival, and are used to guide appropriate treatment strategies. Lymphovascular invasion (LBVI), including lymphatic (LVI) and blood (BVI) vessel invasion, has been reported to be prognostic and merit accurate evaluation, particularly in patients with node negative tumours who might benefit from adjuvant chemotherapy. There is a lack of standard assessment and agreement on distinguishing LVI from BVI despite the major challenges in the field. A systematic review of the literatures, examining methods of detection and the prognostic significance of LBVI, LVI and BVI, was carried out. The majority of studies used haematoxylin and eosin (H&E) and classical histochemistry to identify LVI and BVI. Only few recent studies used immunohistochemistry (IHC) staining of the endothelium lining lymphatic and blood vessels, and were able to show clear differences between LVI and BVI. The prognostic significance of LBVI and LVI was well-documented and strongly associated with aggressive features of breast tumours, while the prognostic value and the optimal detection method of BVI were unclear. Assessment and prognostic value of LBVI on H&E sections (LBVIH&E) was examined and compared to that of LVI and BVI detected using IHC with D2-40 for LVI (LVID2–40) and Factor VIII for BVI (BVIFVIII) in patients with breast cancer including node negative and triple negative patients (n=360). LBVIH&E, LVID2–40 and BVIFVIII were present in 102 (28%), 127 (35%) and 59 (16%) patients respectively. In node negative patients (206), LBVIH&E, LVID2–40 and BVIFVIII were present in 41 (20%), 53 (26%) and 21 (10%) respectively. In triple negative patients (102), LBVIH&E, LVID2–40 and BVIFVIII were present in 35 (29%), 36 (35%) and 14 (14%) respectively. LBVIH&E, LVID2–40 and BVIFVIII were all significantly associated with tumour recurrence in all cohorts. On multivariate survival analysis, only LVID2–40 and BVIFVIII were independent predictors of cancer specific survival (CSS) in the whole cohort (P=0.022 and P<0.001 respectively), node negative (P=0.008 and P=0.001 respectively) and triple negative patients (P=0.014 and P<0.001 respectively). Assessment of LVI and BVI by IHC, using D2-40 and Factor VIII, improves prediction of outcome in patients with node negative and triple negative breast cancer and was superior to the conventional detection method. Breast cancer is recognised as a complex molecular disease and histologically identical tumours may have highly variable outcomes, including different responses to therapy. Therefore, there is a compelling need for new prognostic and predictive markers helpful of selecting patients at risk and patients with aggressive diseases who might benefit from adjuvant and targeted therapy. It is increasingly recognised that the development and progression of human breast cancer is not only determined by genetically abnormal cells, but also dependent on complex interactions between malignant cells and the surrounding microenvironment. This has led to reconsider the features of tumour microenvironment as potential predictive and prognostic markers. Among these markers, tumour stroma percentage (TSP) and tumour budding, as well as local tumour inflammatory infiltrate have received recent attention. In particular, the local environment of cytokines, proteases, angiogenic and growth factors secreted by inflammatory cells and stromal fibroblasts has identified crucial roles in facilitating tumour growth, and metastasis of cancer cells through lymphatic and/or blood vessel invasion. This might help understand the underlying process promoting tumour invasion into these vessels. An increase in the proportion of tumour stroma and an increase in the dissociation of tumour cells have been associated with poorer survival in a number of solid tumours, including breast cancer. However, the interrelationship between these variables and other features of the tumour microenvironment in different subgroups of breast cancer are not clear. Also, whether their prognostic values are independent of other components of the tumour microenvironment have yet to be identified. Therefore, the relationship between TSP, clinicopathological characteristics and outcome in patients with invasive ductal breast cancer, in particular node negative and triple negative disease was examined in patients with invasive ductal breast cancer (n=361). The TSP was assessed on the haematoxylin and eosin-stained tissue sections. With a cut-off value of 50% TSP, patients with ≤50% stroma were classified as the low-TSP group and those with >50% stroma were classified as the high-TSP group. A total of 109 (30%) patients had high TSP. Patients with high TSP were old age (P=0.035), had involved lymph node (P=0.049), Her-2 positive tumours (P=0.029), low-grade peri-tumoural inflammatory infiltrate (P=0.034), low CD68+ macrophage infiltrate (P<0.001), low CD4+ (P=0.023) and low CD8+ T-lymphocytes infiltrate (P=0.017), tumour recurrence (P=0.015) and shorter CSS (P<0.001). In node negative patients (n=207), high TSP was associated with low CD68+ macrophage infiltrate (P=0.001), low CD4+ (P=0.040) and low CD8+ T-lymphocytes infiltrate (P=0.016) and shorter CSS (P=0.005). In triple negative patients (n=103), high TSP was associated with increased tumour size (P=0.017) high tumour grade (P=0.014), low CD8+ T-lymphocytes infiltrate (P=0.048) and shorter CSS (P=0.041). The 15-year cancer specific survival rate was 79% vs 21% in the low-TSP group vs high-TSP group. On multivariate survival analysis, a high TSP was associated with reduced CSS in the whole cohort (P=0.007), node negative patients (P=0.005) and those who received systemic adjuvant therapy (P=0.016), independent of other pathological characteristics including local host inflammatory responses. Therefore, a high TSP in invasive ductal breast cancer was associated with recurrence and poorer long-term survival. The inverse relation with the tumour inflammatory infiltrate highlights the importance of the amount of tumour stroma on immunological response in patients with invasive ductal breast cancer. Implementing this simple and reproducible parameter in routine pathological examination may help optimise risk stratification in patients with breast cancer. Similarly, the relationship between tumour budding, clinicopathological characteristics and outcome was examined in patients with invasive ductal breast cancer (n=474), using routine pathological sections. Tumour budding was associated with several adverse pathological characteristics, including positive lymph node (P=0.009), presence of LVI (P<0.001), and high TSP (P=0.001) and low-grade general peri-tumural inflammatory infiltrative (P=0.002). In node negative patients, a high tumour budding was associated with presence of LVI (P<0.001) and low-grade general peri-tumural inflammatory infiltrative (P=0.038). On multivariate survival analysis, tumour budding was associated with reduced CSS (P=0.001), independent of nodal status, tumour necrosis, CD8+ and CD138+ inflammatory cells infiltrate, LVI, BVI and TSP. Furthermore, tumour budding was independently associated with reduced CSS in node negative patients (P=0.004) and in those who have low TSP (P=0.003) and high-grade peri-tumoural inflammatory infiltrative (P=0.012). A high tumour budding was significantly associated with shorter CSS in luminal B and triple negative breast cancer subtypes (all P<0.001). Therefore, tumour budding was a significant predictor of poor survival in patients with invasive ductal breast cancer, independent of adverse pathological characteristics and components of tumour microenvironment. These results suggest that tumour budding may promote disease progression through a direct effect on local and distant invasion into lymph nodes and lymphatic vessels. Therefore, detection of tumour buds at the stroma invasive front might therefore represent a morphologic link between tumour progression, lymphatic invasion, spread of tumour cells to regional lymph nodes, and the establishment of metastatic dissemination. Given the potential importance of the tumour microenvironment, the characterisation of intracellular signalling pathways is important in the tumour microenvironment and is of considerable interest. One plausible signalling molecule that links tumour stroma, inflammatory cell infiltrate and tumour budding is the signal transducer and activator of transcription (STAT). The relationship between total and phosphorylated STAT1 (ph-STAT1), and total and ph-STAT3 tumour cell expression, components of tumour microenvironment and survival in patients with invasive ductal breast cancer was examined. IHC of total and ph-STAT1/STAT3 was performed on tissue microarray of 384 breast cancer specimens. Cellular STAT1 and cellular STAT3 expression at both cytoplasmic and nuclear locations were combined and identified as STAT1/STAT3 tumour cell expression. These results were then related to CSS and phenotypic features of the tumour and host. A high ph-STAT1 and a high ph-STAT3 tumour cell expression was associated with increased ER (P=0.001 and P<0.001 respectively) and PR (all P<0.05), reduced tumour grade (P=0.015 and P<0.001 respectively) and necrosis (all P=0.001). Ph-STAT1 was associated with increased general peri-tumoural inflammatory infiltrate (P=0.007) and ph-STAT3 was associated with lower CD4+ T-lymphocyte infiltrate (P=0.024). On multivariate survival analysis, including both ph-STAT1 and ph-STAT3 tumour cell expression, only high ph-STAT3 tumour cell expression was significantly associated with improved CSS (P=0.010) independent of other tumour and host-based factors. In patients with high necrosis grade, high ph-STAT3 tumour cell expression was independent predictor of improved CSS (P=0.021). Ph-STAT1 and ph-STAT3 were also significantly associated with improved cancer specific survival in luminal A and B subtypes. STAT1 and STAT3 tumour cell expression appeared to be an important determinant of favourable outcome in patients with invasive ductal breast cancer. The present results suggest that STATs may affect disease outcome through direct impact on tumour cells, and the surrounding microenvironment. The above observations of the present thesis point to the importance of the tumour microenvironment in promoting tumour budding, LVI and BVI. The observations from STATs work may suggest that an important driving mechanism for the above associations is the presence of tumour necrosis, probably secondary to hypoxia. Further work is needed to examine the interaction of other molecular pathways involved in the tumour microenvironment, such as HIF and NFkB in patients with invasive ductal breast cancer.

Specificity in transforming growth factor β-induced transcription of the plasminogen activator inhibitor-1 gene: Interactions of promoter DNA, transcription factor μE3, and Smad proteins

Transforming growth factor β (TGF-β) regulates a broad range of biological processes, including cell growth, development, differentiation, and immunity. TGF-β signals through its cell surface receptor serine kinases that phosphorylate Smad2 or Smad3 proteins. Because Smad3 and its partner Smad4 bind to only 4-bp Smad binding elements (SBEs) in DNA, a central question is how specificity of TGF-β-induced transcription is achieved. We show that Smad3 selectively binds to two of the three SBEs in PE2.1, a TGF-β-inducible fragment of the plasminogen activator inhibitor-1 promoter, to mediate TGF-β-induced transcription; moreover, a precise 3-bp spacer between one SBE and the E-box, a binding site for transcription factor μE3 (TFE3), is essential for TGF-β-induced transcription. Whereas an isolated Smad3 MH1 domain binds to TFE3, TGF-β receptor-mediated phosphorylation of full-length Smad3 enhances its binding to TFE3. Together, these studies elucidate an important mechanism for specificity in TGF-β-induced transcription of the plasminogen activator inhibitor-1 gene.

Repressor- and activator-type ethylene response factors functioning in jasmonate signaling and disease resistance identified via a genome-wide screen of Arabidopsis transcription factor gene expression

To identify transcription factors (TFs) involved in jasmonate (JA) signaling and plant defense, we screened 1,534 Arabidopsis (Arabidopsis thaliana) TFs by real-time quantitative reverse transcription-PCR for their altered transcript at 6 h following either methyl JA treatment or inoculation with the incompatible pathogen Alternaria brassicicola. We identified 134 TFs that showed a significant change in expression, including many APETALA2/ethylene response factor (AP2/ERF), MYB, WRKY, and NACTF genes with unknown functions. Twenty TF genes were induced by both the pathogen and methyl JA and these included 10 members of the AP2/ERF TF family, primarily from the B1a and B3 subclusters. Functional analysis of the B1a TF AtERF4 revealed that AtERF4 acts as a novel negative regulator of JA-responsive defense gene expression and resistance to the necrotrophic fungal pathogen Fusarium oxysporum and antagonizes JA inhibition of root elongation. In contrast, functional analysis of the B3 TF AtERF2 showed that AtERF2 is a positive regulator of JA-responsive defense genes and resistance to F. oxysporum and enhances JA inhibition of root elongation. Our results suggest that plants coordinately express multiple repressor-and activator-type AP2/ERFs during pathogen challenge to modulate defense gene expression and disease resistance.

Transcription of Human Resistin Gene Involves an Interaction of Sp1 with Peroxisome Proliferator-Activating Receptor Gamma (PPAR gamma)

Background: Resistin is a cysteine rich protein, mainly expressed and secreted by circulating human mononuclear cells. While several factors responsible for transcription of mouse resistin gene have been identified, not much is known about the factors responsible for the differential expression of human resistin.Methodology/Principal Finding: We show that the minimal promoter of human resistin lies within similar to 80 bp sequence upstream of the transcriptional start site (-240) whereas binding sites for cRel, CCAAT enhancer binding protein alpha (C/EBP-alpha), activating transcription factor 2 (ATF-2) and activator protein 1 (AP-1) transcription factors, important for induced expression, are present within sequences up to -619. Specificity Protein 1(Sp1) binding site (-276 to -295) is also present and an interaction of Sp1 with peroxisome proliferator activating receptor gamma (PPAR gamma) is necessary for constitutive expression in U937 cells. Indeed co-immunoprecipitation assay demonstrated a direct physical interaction of Sp1 with PPAR gamma in whole cell extracts of U937 cells. Phorbol myristate acetate (PMA) upregulated the expression of resistin mRNA in U937 cells by increasing the recruitment of Sp1, ATF-2 and PPAR gamma on the resistin gene promoter. Furthermore, PMA stimulation of U937 cells resulted in the disruption of Sp1 and PPAR gamma interaction. Chromatin immunoprecipitation (ChIP) assay confirmed the recruitment of transcription factors phospho ATF-2, Sp1, Sp3, PPAR gamma, chromatin modifier histone deacetylase 1 (HDAC1) and the acetylated form of histone H3 but not cRel, C/EBP-alpha and phospho c-Jun during resistingene transcription.Conclusion: Our findings suggest a complex interplay of Sp1 and PPAR gamma along with other transcription factors that drives the expression of resistin in human monocytic U937 cells.

Elucidation of the interactions between the transcription factor E2A and the transcriptional co-activator CBP/p300

E2A is a transcription factor that plays a particularly critical role in lymphopoiesis. The chromosomal translocation 1;19, disrupts the E2A gene and results in the expression of the fusion oncoprotein E2A-PBX1, which is implicated in acute lymphoblastic leukemia. Both E2A and E2A-PBX1 contain two activation domains, AD1 and AD2, which comprise conserved ΦxxΦΦ motifs where Φ denotes a hydrophobic amino acid. These domains function to recruit transcriptional co-activators and repressors, including the histone acetyl transferase CREB binding protein (CBP) and its paralog p300. The PCET motif within E2A AD1 interacts with the KIX domain of CBP/p300, the disruption of which abrogates the transcriptional activation by E2A and the transformative properties of E2A-PBX1. The generation of a peptide-based inhibitor targeting the PCET:KIX interaction would serve useful in further assessing the role of E2A and E2A-PBX1 in lymphopoiesis and leukemogenesis. An interaction between E2A AD2 and the KIX domain has also been recently identified, and the TAZ domains of CBP/p300 have been shown to interact with several transcription factors that contain ΦxxΦΦ motifs. Thus the design of an inhibitor of the E2A:CBP/p300 interaction requires the full complement of interactions between E2A and the various domains of CBP/p300 to be elucidated. Here, we have used nuclear magnetic resonance (NMR) spectroscopy to determine that AD2 interacts with KIX at the same site as PCET, which indicates that the E2A:KIX interaction can be disrupted by targeting a single binding site. Using an iterative synthetic peptide microarray approach, a peptide with the sequence DKELQDLLDFSLQY was derived from PCET to interact with KIX with higher affinity than the wild type sequence. This peptide now serves as a lead molecule for further development as an inhibitor of the E2A:CBP/p300 interaction. Fluorescence anisotropy, peptide microarray technology, and isothermal titration calorimetry were employed to characterize interactions between both TAZ domains of CBP/p300 and the PCET motif and AD2 of E2A. Alanine substitution of residues within PCET demonstrated that the ΦxxΦΦ motif is a key mediator of these interactions, analogous to the PCET:KIX interaction. These findings now inform future work to establish possible physiological roles for the E2A:TAZ1 and E2A:TAZ2 interactions.

Elevated beta1,4-galactosyltransferase I in highly metastatic human lung cancer cells. Identification of E1AF as important transcription activator.

The elevated levels of beta1,4-galactosyltransferase I (GalT I; EC 2.4.1.38) are detected in highly metastatic lung cancer PGBE1 cells compared with its less metastatic partner PGLH7 cells. Decreasing the GalT I surface expression by small interfering RNA or interfering with the surface of GalT I function by mutation inhibited cell adhesion on laminin, the invasive potential in vitro, and tyrosine phosphorylation of focal adhesion kinase. The mechanism by which GalT I activity is up-regulated in highly metastatic cells remains unclear. To investigate the regulation of GalT I expression, we cloned the 5'-region flanking the transcription start point of the GalT I gene (-1653 to +52). Cotransfection of the GalT I promoter/luciferase reporter and the Ets family protein E1AF expression plasmid increased the luciferase reporter activity in a dose-dependent manner. By deletion and mutation analyses, we identified an Ets-binding site between nucleotides -205 and -200 in the GalT I promoter that was critical for responsiveness to E1AF. It was identified that E1AF could bind to and activate the GalT I promoter by electrophoretic mobility shift assay in PGLH7 cells and COS1 cells. A stronger affinity of E1AF for DNA has contributed to the elevated expression of GalT I in PGBE1 cells. Stable transfection of the E1AF expression plasmid resulted in increased GalT I expression in PGLH7 cells, and stable transfectants migrated faster than control cells. Meanwhile, the content of the beta1,4-Gal branch on the cell surface was increased in stably transfected PGLH7 cells. GalT I expression can also be induced by epidermal growth factor and dominant active Ras, JNK1, and ERK1. These data suggest an essential role for E1AF in the activation of the human GalT I gene in highly metastatic lung cancer cells.

Calmodulin-binding transcription activator 1 (CAMTA1) alleles predispose human episodic memory performance

Little is known about the genes and proteins involved in the process of human memory. To identify genetic factors related to human episodic memory performance, we conducted an ultra-high-density genome-wide screen at > 500 000 single nucleotide polymorphisms (SNPs) in a sample of normal young adults stratified for performance on an episodic recall memory test. Analysis of this data identified SNPs within the calmodulin-binding transcription activator 1 (CAMTA1) gene that were significantly associated with memory performance. A follow up study, focused on the CAMTA1 locus in an independent cohort consisting of cognitively normal young adults, singled out SNP rs4908449 with a P-value of 0.0002 as the most significant associated SNP in the region. These validated genetic findings were further supported by the identification of CAMTA1 transcript enrichment in memory-related human brain regions and through a functional magnetic resonance imaging experiment on individuals matched for memory performance that identified CAMTA1 allele-specific upregulation of medial temporal lobe brain activity in those individuals harboring the 'at-risk' allele for poorer memory performance. The CAMTA1 locus encodes a purported transcription factor that interfaces with the calcium-calmodulin system of the cell to alter gene expression patterns. Our validated genomic and functional biological findings described herein suggest a role for CAMTA1 in human episodic memory.

Role of the transcription factor activator protein-2alpha in prostate carcinogenesis

Activator protein 2α (AP-2) is a transcription factor known to play a crucial role in the progression of malignant melanoma, colorectal carcinoma, and breast cancer. Several AP-2 target genes are known to be deregulated in prostate cancer, therefore, we hypothesize that loss AP-2 expression plays a causal role in prostate carcinogenesis. Immunofluorescent staining for AP-2 of 30 radical prostatectomy specimens demonstrated that while AP-2 was highly expressed in normal prostate epithelium, its expression was lost in most cases of high grade prostatic intraepithelial neoplasia (PIN), and all cases of prostate cancer studied. Additional analyses demonstrated that AP-2 was associated with normal luminal differentiation and it was not expressed in the basal cell layer. In cell lines, AP-2 was strongly expressed in immortalized normal prostate epithelial cells, whereas low expression was observed in the LNCaP, LNCaP-LN3, and PC3M-LN4 prostate cancer cell lines. Transfection of the highly tumorigenic and metastatic cell line PC3M-LN4 with the AP-2 gene significantly decreased tumor growth in the prostate of nude mice (p = 0.032) and inhibited metastases to the lymph nodes. Moreover, transfection of the low tumorigenic, low metastatic cell line LNCaP-LN3 with full length AP-2; resulted in complete inhibition of tumor incidence in the AP-2 transfectants (0/19) vs. neo control (10/16). A potential mechanism for this loss of tumorigenicity was the modulation of gene expression in prostate cancer cells that mimicked the normal phenotype. Analysis of differential expression between neo control- and AP-2-transfected cells in vitro and in tumors demonstrated low VEGF expression in AP-2 transfectants. We further demonstrated that AP-2 acted as a transcriptional repressor of the VEGF promoter by binding to a GC-rich region located between −88 and −66. This region contains an AP-2 consensus element overlapping two Sp1 consensus elements. We found that Sp3 and AP-2 bound to this region in a mutually exclusive manner to promote activation or repression. Increased VEGF expression has been observed in high grade PIN and in prostate cancer. Here we provide evidence that this early molecular change could be a result of loss of AP-2 expression in the prostatic epithelium. ^

DNase I-hypersensitive sites I and II of the human growth hormone locus control region are a major developmental activator of somatotrope gene expression

High-level expression of the human growth hormone (hGH) gene is limited to somatotrope and lactosomatotrope cells of the anterior pituitary. We previously identified a locus control region (LCR) for the hGH gene composed of four tissue-specific DNase I-hypersensitive sites (HS) located between −14.6 kb and −32 kb 5′ to the hGH transcription start site that is responsible for establishing a physiologically regulated chromatin domain for hGH transgene expression in mouse pituitary. In the present study we demonstrated that the LCR mediates somatotrope and lactosomatotrope restriction on an otherwise weakly and diffusely expressed hGH transgene. The subregion of the LCR containing the two pituitary-specific HS, HSI and HSII (−14.6 to −16.2 kb relative to the hGH promoter and denoted HSI,II), was found to be sufficient for mediating somatotrope and lactosomatotrope restriction, for appropriately timed induction of hGH transgene expression between embryonic days 15.5 and 16.5, and for selective extinction of hGH expression in mature lactotropes. When studied by cell transfection, the HSI,II fragment selectively enhanced transcription in a presomatotrope-derived cell line, although at levels (2- to 3-fold) well below that seen in vivo. The LCR activity of the HSI,II element was therefore localized by scoring transgene expression in fetal founder pituitaries at embryonic day 18.5. The data from these studies indicated that a 404-bp segment of the HSI,II region encodes a critical subset of LCR functions, including the establishment of a productive chromatin environment, cell-specific restriction and enhancement of expression, and appropriately timed induction of the hGH transgene during embryonic development.