THE ROLE OF TAK1 IN PANCREATIC CANCER DEVELOPMENT

Pancreatic cancer is the fourth leading cause of cancer-related mortality in the United States and the fifth leading cause of cancer-related mortality worldwide. Pancreatic cancer is a big challenge in large due to the lack of early symptoms. In addition, drug resistance is a major obstacle to the success of chemotherapy in pancreatic cancer. The underlying mechanism of drug resistance in human pancreatic cancers is not well understood. Better understanding of the mechanism of molecular pathways in human pancreatic cancers can help to identify the novel therapeutic target candidates, and develop the new preventive and clinic strategies to improve patient survival. We discovered that TAK1 is overexpressed in pancreatic cancer cell lines and patient tumor tissues. We demonstrated that the elevated activity of TAK1 is caused by its binding partner TAB1. Knocking down of TAK1 in pancreatic cancer cells with RNAi technique resulted in cell apoptosis and significantly reduces the size of tumors in mice and made a chemotherapy drug more potent. Targeting the kinase activity of TAK1 with the selective inhibitor LY2610956 strongly synergized in vitro with the antitumor activity of gemcitabine, oxaliplatin, or irinotecan on pancreatic cancer cells. These findings highlighted that TAK1 could be a potential therapeutic target for pancreatic cancer. We also demonstrated that TAK activity is regulated by its binding protein TAB1. We defined a minimum TAB1 sequence which is required and sufficient for TAK1 kinase activity. We created a recombinant TAK1-TAB1 C68 fusion form which has highly kinase activity. This active form could is used for screening TAK1 inhibitors. In addition, several posttranslational modifications were identified in our study. The acetylation of lysine 158 on TAK1 is required for kinase activity. This site is conserved throughout all of kinase. Our findings may reveal a new mechanism by which kinase activity is regulated.

THE ROLE OF E2F1 IN THE RESPONSE TO DNA DOUBLE STRAND BREAKS

The importance of E2F transcription factors in the processes of proliferation and apoptosis are well established. E2F1, but not other E2F family members, is also phosphorylated and stabilized in response to various forms of DNA damage to regulate the expression of cell cycle and pro-apoptotic genes. E2F1 also relocalizes and forms foci at sites of DNA double-strand breaks but the function of E2F1 at sites of damage is still unknown. Here I reveal that E2F1 deficiency leads to increased spontaneous DNA break and impaired recovery following exposure to ionizing radiation. In response to DNA double-strand breaks, NBS1 phosphorylation and foci formation are defective in cells lacking E2F1, but NBS1 expression levels are unaffected. Moreover, it was observed that an association between NBS1 and E2F1 is increased in response to DNA damage, suggesting that E2F1 may promote NBS1 foci formation through a direct or indirect interaction at sites of DNA breaks. E2F1 deficient cells also display impaired foci formation of RPA and Rad51, which suggests a defect in DNA end resection and formation of single-stranded DNA at DNA double-strand breaks. I also found E2F1 status affects foci formation of the histone acetyltransferase GCN5 in response to DNA double-strand breaks. E2F1 is phosphorylated at serine 31 (serine 29 in mouse) by the ATM kinase as part of the DNA damage response. To investigate the importance of this event, our lab developed an E2F1 serine 29 mutant mouse model. I find that E2F1 serine 29 mutant cells show loss of E2F1 foci formation in response to DNA double-strand breaks. Furthermore, DNA repair and NBS1 foci formation are impaired in E2f1S29A/S29A cells. Taken together, my results indicate novel roles for E2F1 in the DNA damage response, which may directly promote DNA repair and genome maintenance.

The Role of Cortisol in the Cycle of Violence

Child abuse and neglect are universal risk factors for delinquency, violence and aggression; this phenomenon is known as the cycle of violence. Despite a wide body of research demonstrating this phenomenon, the processes which mediate this relationship remain largely unknown. One potentially relevant result of abuse and neglect may be disruptions in the development of the body’s stress response, specifically the function of the Hypothalamic-Pituitary-Adrenal (HPA) axis. The HPA-axis, and its end-product, cortisol, may play a role in regulating aggressive behavior, but this function may be disrupted following abuse and neglect. Another risk factor for aggression, psychopathy, may mediate the cycle of violence or independently contribute to aggressive behavior. This study examined the relationship between child abuse and neglect, HPA-axis function, psychopathy and aggression. History of abuse was measured using a self-report questionnaire, the Childhood Trauma Questionnaire. Using a within-subject, placebo-controlled, counter-balanced dosing design, 67 adults were given an acute dose of 20mg cortisol as a challenge to the HPA-axis. Following dosing, measures of cortisol response were obtained through saliva samples, and state-aggressive behavior was measured by a laboratory task, the Point-Subtraction Aggression Paradigm (PSAP). Basal measures of cortisol were obtained prior to dosing. Psychopathy and a trait-measure of aggression were assessed through self-report questionnaires. PSAP data and trait-aggression scores were normalized and summed for an overall aggression score. Linear regression analyses indicated that a history of abuse and neglect robustly predicted aggression, supporting the cycle of violence hypothesis. Further, abuse and neglect predicted a diminished HPA-axis response to the cortisol challenge. Although a diminished HPA-axis response significantly predicted increased aggression, mediation analysis revealed that HPA-axis reactivity did not mediate a significant portion of the effect of abuse and neglect on aggression. However, HPA-axis reactivity did mediate part of the effect, indicating that HPA-axis function may be a factor in the cycle of violence. Psychopathy robustly predicted increased aggression. Although the results indicate that cortisol, psychopathy and HPA-axis function are involved in the cycle of violence, further research is required to better understand the complex interaction of these factors.

Studies on the role of BCR in Philadelphia chromosome-positive leukemias

It is well established that the chimeric Bcr-Abl oncoprotein resulting from fusing 3$\sp\prime$ ABL sequences on chromosome 9 to 5$\sp\prime$ BCR sequences on chromosome 22 is the primary cause of Philadelphia chromosome-positive (Ph$\sp1$) leukemias. Although it is clear that the cis-Bcr sequence present within Bcr-Abl is able to activate the tyrosine kinase activity and F-actin binding capacity of Bcr-Abl which is critical for the transforming ability of BCR-ABL, the biological role of normal BCR gene product (P160 BCR) remains largely unknown. The previous finding by our lab that P160 BCR forms stable complexes with Bcr-Abl oncoprotein in Ph$\sp1$-positive leukemic cells implicated P160 BCR in the pathogenesis of Ph$\sp1$-positive leukemias. Here, we demonstrated that P160 BCR physically interacts with P210 BCR-ABL and become tyrosine phosphorylated when co-expressed with P210 BCR-ABL in COS1 cells while no tyrosine phosphorylation of P160 BCR can be detected when it is expressed alone. The results suggest that P160 BCR is a target for the Bcr-Abl tyrosine kinase. Although we were unable to detect stable physical interaction between P160 BCR and P145 c-ABL (Ib) in COS1 cells overexpressing both proteins, P160 BCR was phosphorylated on tyrosine residues when co-expressed with activated tyrosine kinase of P145 c-ABL (Ib). In addition, studies of tyrosine phosphorylation of BCR deletion mutants and 2-dimensional tryptic mapping of in vitro phosphorylated wild type and mutant (tyrosine to phenylalanine) Bcr-Abl indicated that tyrosine 177, 283 and 360 of Bcr represent some of the phosphorylation sites. Even though the significance of tyrosine phosphorylation of residues 283 and 360 of Bcr has not been determined, tyrosine phosphorylation of residue 177 within Bcr-Abl has been reported to be critical for its interaction with Grb2 molecule and subsequent activation of Ras signaling pathway. Here, we further demonstrated that tyrosine 177 phosphorylated P160 BCR is also able to bind to Grb2 molecule suggesting the role of P160 BCR in the Ras signaling pathway.^ Surprisingly, using 3$\sp\prime$ BCR antisense oligonucleotide to reduce the expression of P160 BCR without interfering with the expression of BCR-ABL resulted in increased growth or survival of B15 cells and M3.16 cells expressing either P185 BCR-ABL or P210 BCR-ABL respectively. The results provided strong arguments that P160 BCR may function as a negative regulator for cell growth.^ Considering all these results, we hypothesize that P160 BCR negatively regulate cell growth and tyrosine phosphorylation of P160 BCR turns off its growth suppressor function and turns on its growth stimulatory function. We further speculate that Bcr-Abl oncoprotein in leukemia cells stably interacts with and constitutively phosphorylates portions of P160 BCR converting it into a growth stimulatory state. In normal cells, the growth suppressor effects of P160 BCR could only be transiently and conditionally switched to growth stimulatory action by a strictly regulated cellular tyrosine kinase such as c-ABL. The model will be further discussed in the text. ^

The role of menaquinone in the nitrate reductase complex

Membrane bound, respiratory nitrate reductase in Escherichia coli is composed of three subunits, αβγ. The active complex is anchored to the membrane by membrane-integrated γ subunit and can reduce nitrate to nitrite with membrane quinones, (ubiquinone or menaquinone) as physiological electron donors. The transfer of electrons through the complex is thought to involve the sequence: membrane quinols → b-type hemes (γ subunit) → Fe-S centers (β subunit) → molybdopterin (α subunit) → nitrate. The enzyme can be assayed with the artificial electron donor reduced methyl viologen (MVH) which transfers electrons directly to the molybdopterin cofactor. These studies have focused on the possible role of protein-bound menaquinone in the structure and function of this multisubunit complex. ^ Nitrate reductase was purified as two distinct forms; after solubilization of membrane proteins with detergents, purification rendered an αβγ complex (holoenzyme) which catalyzes nitrate reduction with MVH or the quinols analogs, menadiol and duroquinol, as electron donors. Alternatively, heat-treatment of the membranes in the absence of detergents and subsequent purification of the active enzyme produced an αβ complex, which reduces nitrate only with MVH as electron donor. The active αβ dimer was also separated from γ subunit by heat treatment of the holoenzyme. ^ Menaquinone-9 was isolated directly from the purified αβ complex, and identified by mass spectrometry. Based on the composition of the membrane quinone pool, it was concluded that menaquinone-9 is sequestered from the membrane pool in a specifically protein-bound form. ^ The role of the bound menaquinone in the structure-function of nitrate reductase was also investigated, along with its participation in UV-light inactivation of the enzyme. Menaquinone-depleted nitrate reductase from a menaquinone deficient mutant retained activity with all electron donors and it remained sensitive to UV inactivation. However, the MVH-nitrate reductase activity and the rate of UV inactivation of the enzyme were significantly reduced and the optical properties of the enzyme were modified by the absence of the bound menaquinone-9. ^ Menaquinone-9 is not absolutely required for electron transfer in nitrate reductase but it appears to be specifically-bound during assembly of the complex and to enhance the transfer of electrons through the complex. The possible plasticity of the functional electron transfer pathway in nitrate reductase is discussed. ^

ROLE OF GLUTATHIONE IN FORMALDEHYDE METABOLISM AND TOXICITY

Glutathione (GSH) is involved in the detoxication of numerous chemicals exogenously exposed or endogenously generated. Exposure to these agents cause depletion of cellular GSH rendering these cells more susceptible to the toxic action of these same agents. Formaldehyde (CH(,2)O) was found to deplete cellular GSH, presumably by the formation of the GSH-CH(,2)O complex, S-hydroxymethylglutathione, and its rapid extrusion into the extracellular medium.^ The metabolism and toxicity of CH(,2)O were determined to be dependent upon cellular GSH in vitro and in vivo. The rate of CH(,2)O oxidation decreased and the extent of toxicity increased when isolated rat hepatocytes or strain A/J mice were pretreated with the GSH-depleting agent, diethyl maleate (DEM). Additional experiments were designed to further study the role GSH plays in detoxication using isolated rat hepatocytes.^ L-Methionine protected against the extent of lipid peroxidation and leakage of the cytosolic enzyme, lactate dehydrogenase (LDH), caused by CH(,2)O in DEM-pretreated hepatocytes, further supporting the protective role of GSH against cellular toxicity. The antioxidants, ascorbate, butylated hydroxytoluene, and (alpha)-tocopherol, were all protective against the extent of lipid peroxidation and leakage of LDH in isolated rat hepatocytes. Whereas L-methionine may be protective by increasing the cellular concentration of GSH which is used to detoxify free radicals or by facilitating the rate of CH(,2)O oxidation, the antioxidant, ascorbate, was protective without altering the rate of CH(,2)O oxidation or increasing cellular GSH levels. These results suggest that the free radical-mediated toxicity caused by CH(,2)O in DEM-pretreated hepatocytes is due to the further depletion of GSH by CH(,2)O and not to increased CH(,2)O persistence. How this further depletion in GSH by CH(,2)O in DEM-pretreated hepatocytes results in lipid peroxidation and cell death was further investigated.^ The further decrease in GSH caused by CH(,2)O in DEM-pretreated hepatocytes, suspected of stimulating lipid peroxidation and cell death, was found not to be due to depletion of mitochondrial GSH but to depletion of protein sulfhydryl groups. In addition, cellular toxicity appears more closely correlated with depletion of protein sulfhydryl groups than with an increase in cytosolic free Ca('2+). The combination of CH(,2)O and DEM may be a useful tool in identifying these critical sulfhydryl-protein(s) and to further understand the role GSH plays in detoxication. ^

How nice is good for patients and for therapy outcome? The role of confrontation in the process of psychotherapy

It is well established that the therapeutic relationship contributes about as much to therapy outcome as 'technical' intervention. Furthermore, it follows clear prescriptive concepts in the same manner as technical interventions do. 'Motive Oriented Therapeutic Relationship' is such a concept for establishing a solid basis for whatever therapeutic work the patients' problems require (Grawe, 1980, 1992; Caspar, 1996). Yet, the therapeutic relationship doesn't explain everything because other factors play a significant role too. Previous studies showed that outcome is clearly better when therapists achieved a generally high quality of a therapeutic relationship when they did not shy away from possibly threatening interventions such as confrontations. This ratio of a fruitful alliance and marginally present confrontations in the same session also showed significant correlations with patient's assessment of alliance and progress in therapy (Figlioli et al., 2009).Aim: The current state of research in the field does not give any answers to questions like how good and bad confrontations can be characterized or what role does the intensity, respectively frequency of confrontations play in the process of psychotherapy. Methods: A sample of 80 therapies of 3 sessions each representing either good or bad outcome was judged moment by moment by independent raters if and how therapists used confrontative interventions. Results: Preliminary analyses show that successful confrontations are explicitly uttered, short but intense, related to important patients goals in therapy and embedded in prior complementarity. Discussion: The results will be discussed in terms of their implications for the clinical daily work.

How nice is good for patients and for therapy outcome? The role of confrontation in the process of psychotherapy

It is well established that the therapeutic relationship contributes about as much to therapy outcome as ‘technical’ intervention. Furthermore, it follows clear prescriptive concepts in the same manner as technical interventions do. ‘Motive Oriented Therapeutic Relationship’ is such a concept for establishing a solid basis for whatever therapeutic work the patients’ problems require (Grawe, 1980, 1992; Caspar, 1996). Yet, the therapeutic relationship doesn’t explain everything because other factors play a significant role too. Previous studies showed that outcome is clearly better when therapists achieved a generally high quality of a therapeutic relationship when they did not shy away from possibly threatening interventions such as confrontations. This ratio of a fruitful alliance and marginally present confrontations in the same session also showed significant correlations with patient’s assessment of alliance and progress in therapy (Figlioli et al., 2009). These findings are also very much in line with Sachse’s metaphor of accumulating, but then also using ‘relationship credits’ and Farrelly’s ‘Provocative Therapy’ (1986), as well as the ‘Intensive Short-Term Dynamic Psychotherapy’ by Davanloo (1980).Aim: The current state of research in the field does not give any answers to questions like how good and bad confrontations can be characterized or what role does the intensity, respectively frequency of confrontations play in the process of psychotherapy.Methods: A sample of 80 therapies of 3 sessions each representing either good or bad outcome was judged moment by moment by independent raters if and how therapists used confrontative interventions. Results / Discussion: The results will be discussed in terms of their implications for the clinical daily work. Preliminary analyses show that successful confrontations are explicitly uttered, short but intense, related to important patients goals in therapy and embedded in prior complementarity.

Potential role of CYP2D6 in the central nervous system

1. Cytochrome P450 2D6 (CYP2D6) is a pivotal enzyme responsible for a major drug oxidation polymorphism in human populations. Distribution of CYP2D6 in brain and its role in serotonin metabolism suggest that CYP2D6 may have a function in the central nervous system. 2. To establish an efficient and accurate platform for the study of CYP2D6 in vivo, a human CYP2D6 (Tg-2D6) model was generated by transgenesis in wild-type (WT) C57BL/6 mice using a P1 phage artificial chromosome clone containing the complete human CYP2D locus, including the CYP2D6 gene and 5'- and 3'-flanking sequences. 3. Human CYP2D6 was expressed not only in the liver but also in the brain. The abundance of serotonin and 5-hydroxyindoleacetic acid in brain of Tg-2D6 is higher than in WT mice, either basal levels or after harmaline induction. Metabolomics of brain homogenate and cerebrospinal fluid revealed a significant up-regulation of L-carnitine, acetyl-L-carnitine, pantothenic acid, 2'-deoxycytidine diphosphate (dCDP), anandamide, N-acetylglucosaminylamine and a down-regulation of stearoyl-L-carnitine in Tg-2D6 mice compared with WT mice. Anxiety tests indicate Tg-2D6 mice have a higher capability to adapt to anxiety. 4. Overall, these findings indicate that the Tg-2D6 mouse model may serve as a valuable in vivo tool to determine CYP2D6-involved neurophysiological metabolism and function.

The role of adipokines in periodontal infection and healing.

Periodontitis is a chronic inflammatory disease of the periodontium, which is caused by pathogenic bacteria in combination with other risk factors. The bacteria induce an immunoinflammatory host response, which can lead to irreversible matrix degradation and bone resorption. Periodontitis can be successfully treated. To achieve regenerative periodontal healing, bioactive molecules, such as enamel matrix derivative (EMD), are applied during periodontal surgery. Recently, it has been shown that obesity is associated with periodontitis and compromised healing after periodontal therapy. The mechanisms underlying these associations are not well understood so far, but adipokines may be a pathomechanistic link. Adipokines are bioactive molecules that are secreted by the adipose tissue, and that regulate insulin sensitivity and energy expenditure, but also inflammatory and healing processes. It has also been demonstrated that visfatin and leptin increase the synthesis of proinflammatory and proteolytic molecules, whereas adiponectin downregulates the production of such mediators in periodontal cells. In addition, visfatin and leptin counteract the beneficial effects of EMD, whereas adiponectin enhances the actions of EMD on periodontal cells. Since visfatin and leptin levels are increased and adiponectin levels are reduced in obesity, these adipokines could be a pathomechanistic link whereby obesity and obesity-related diseases enhance the risk for periodontitis and compromised periodontal healing. Recent studies have also revealed that adipokines, such as visfatin, leptin and adiponectin, are produced in periodontal cells and regulated by periodontopathogenic bacteria. Therefore, adipokines may also represent a mechanism whereby periodontal infections can impact on systemic diseases.

The role of FUS in splicing regulation

Fused in sarcoma (FUS), also called translocated in liposarcoma (TLS), is a ubiquitously expressed DNA/RNA binding protein belonging to the TET family and predominantly localized in the nucleus. FUS is proposed to be involved in various RNA metabolic pathways including transcription regulation, nucleo-cytosolic RNA transport, microRNA processing or pre-mRNA splicing [1]. Mutations in the FUS gene were identified in patients with familial amyotrophic lateral sclerosis (ALS) type 6 and sporadic ALS [2, 3]. ALS, also termed Lou Gehrig's disease, is a fatal adult-onset neurodegenerative disease affecting upper and lower motor neurons in the brain and spinal cord. There is increasing evidence supporting the hypothesis that FUS might play an important role in pre-mRNA splicing regulation. Several splicing factors were identified to associate with FUS including hnRNPA2 and C1/C2 [4], Y-box binding protein 1 (YB-1) [5] and serine arginine (SR) proteins (SC35 and TASR) [6]. Additionally, FUS was identified as a constituent of human spliceosomal complexes [1]. Our recent results indicate that FUS has increased affinity for certain but not all snRNPs of the minor and major spliceosome. Furthermore, in vitro studies revealed that FUS directly interacts with a factor specific for one of those snRNPs. These findings might uncover the molecular mechanism by which FUS regulates splicing and could explain previously observed effects of FUS on the splicing of the adenovirus E1A minigene [7] and changes in splicing caused by ALS associated FUS mutations. [1] Lagier-Tourenne C et al. (2010) Human Molecular Genetics 19:46-64 [2] Kwiatkowski TJ Jr et al. (2009) Science 323:1205-8 [3] Vance C et al. (2009) Science 323:1208-11 [4] Zinser H et al. (1994) Genes Dev 8:2513-26 [5] Chansky, H.A., et al. (2001) Cancer Res. 61: 3586-90. [6] Yang L et al. (1998) J Biol Chem 273:27761-6 [7] Kino Y et al. (2010) Nucleic Acid Research 7:2781-2798