M.Sc. (Biochemistry) / Please refer to full text to view abstract

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Thesis (M. Med. Sc.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 64-72).

Thesis (M.S. in Bio.)--School of Biology, Georgia Institute of Technology, 2004. Directed by Harish Radhakrishna. / Includes bibliographical references (leaves 55-65).

Thesis (Ph. D.)--University of Oregon, 2002. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 84-91). Also available for download via the World Wide Web; free to University of Oregon users.

The c-Jun N-terminal kinases (JNKs) are MAPK family members and are activated by stress, growth factors and cytokines. They are encoded by three separate genes (jnk 1, 2, and 3), spliced alternately creating 10 isoforms. JNK signaling promotes both cell death and cell survival in a stimuli and tissue specic manner and is also implicated in tumorigenesis. Using the Polyoma Virus Middle T Antigen (PyVMT) transgenic mouse model where jnk2 was either expressed or deleted, we found that the PyVMTjnk2-/- tumors expressed higher Epidermal Growth Factor Receptor Substrate 8 (EPS8) mRNA and protein. EPS8 regulates EGFR signaling from Ras to Rac and EGFR tracking via Rab5 and RN-Tre. EPS8 is a prime candidate for connecting the EGFR signaling to actin cytoskeleton remodeling, thus mediating cell migration, a critical step in metastasis. In migration assays, PyVMTjnk2+/+ cells migrated ve fold more than the PyVMTjnk2-/- cells. Re-expression of JNK2[alpha] in the PyVMTjnk2-/- cells rescued this phenotype. Expression of shRNA EPS8 in the PyVMTjnk2-/- cell increased migration in vitro. EPS8 localization at dorsal rues and internalization of EGF-EGFR complexes coincided with JNK2 expression. Expression of shEPS8 in the PyVMTjnk2-/- cells increased EGF internalization suggesting that in absence of JNK2, EPS8 participates in Rab5-RN-Tre complex that inhibits EGFR internalization. Finally, we report that in absence of JNK2, EPS8 protein stability is greatly increased, suggesting that JNK2 is essential for endosomal sorting and degradation of EGFR associated cargo, of which EPS8 is a critical part. In contrast, silencing JNK1 (p46) in 4T1.2 mammary tumor cells, consistently enhanced cell invasion and tumor growth. Tumors derived from orthotopic injection of the 4T1.2shJNK1 expressing cells into the mammary fat pad reached target volume signicantly earlier than non-silencing vector expressing tumors. When injected intravenously, signicantly higher lung metastasis was observed in the 4T1.2shJNK1 group. The more aggressive behavior of 4T1.2shJNK1 tumors was associated with an increase in CCR5 and pAkt as detected by microarray analysis. Taken together, our data suggest that JNK1 suppresses the expression of proteins associated with tumor growth and invasive phenotype, contributing to tumor progression. / text

Due to their role in cellular signaling, mitogen activated protein (MAP) kinases represent targets of pharmaceutical interest. Most MAP kinase inhibitors target the highly conserved ATP binding site. This conservation promotes cross-reactivity and toxicities that may limit their potential as drugs. These drawbacks motivate the search for non-ATP competitive inhibitors with acceptable specificity and potency and also drive efforts to understand MAPK regulation. We applied a virtual screening (VS) workflow to discover novel scaffolds for ATP-independent JNK (C-Jun N-terminal Kinase) inhibitors targeting the JNK-JIP (JNK Interacting Protein) interaction. (-)-Zuonin A was identified as an inhibitor of JNK, exhibiting 100-fold selectivity for the JNKs over other MAP kinases. (-)-Zuonin A was characterized extensively both in vitro and in cell-based assays. The JNK2 isoform has been reported to regulate breast cancer cell migration. Accordingly, we engineered a JNK2-selective peptide inhibitor. Peptides derived from the JIP scaffolds linked to the cell-penetrating peptide TAT are used widely to investigate JNK-mediated signaling events without exibiiting isoform selectivity. Herein, Several JIP-based peptide sequences were designed and tested. A JIP sequence connected through a flexible linker to either the N-terminus of an inverted TAT sequence [mathematical equation], or to a poly-arginine sequence [mathematical equation] enabled the potent inhibition of JNK2 (IC₅₀~90 nM) with 10-fold selectivity over JNK1 and JNK3. Both peptides revealed a potent ability to inhibit the induction of JNK activation and c-Jun phosphorylation in HEK293 cells treated with anisomycin, and inhibited the migration of Polyoma Middle-T Antigen Mammary Tumor (P[subscript y]VMT) cells through the selective inhibition of JNK2. ERK2 dimerization has been reported to regulate its nuclear translocation and signaling. Our analysis using light scattering, analytical ultracentrifugation and NMR provide strong evidence that ERK2 is monomeric under physiological conditions. / text

Ovarian cancer is regarded as the most lethal gynecological malignancy around the world. Despite the advancing medical improvements in both surgery and chemotherapy, the mortality rate did not appear to be reduced. This could be account for the late diagnosis of ovarian cancer until advanced stage. Recently, p-21 activated kinase 4 (PAK4), as a potential significant prognostic marker of ovarian cancer, has been widely studied on its contribution in oncogenesis properties. It was suggested that PAK4 proteins were activated and confer chemoresistance in ovarian cancers. In this study, we hypothesized that the up-regulation of PAK4 in ovarian cancers maybe resulted from mutations and amplification in genomic DNA level. Investigations on PAK4 genetic alterations were carried out. Recurrent mutations were found in the kinase domain of PAK4 in three ovarian cancer cell lines and two clinical samples. Single mutation was found in the exon 3 of PAK4 coding for GTPase binding domain (GTB). Amplifications of PAK4 genomic DNA were also found in four ovarian cancer cell lines. On top of that, dysregulated PAK4 level in chemosensitivity ovarian cancer cell line, A2780s showed PAK4 contribution in protection against apoptosis. Meanwhile PAK4 transfected chemoresistance cell line A2780cp also showed similar effect to PAK4 transfected A2780s. Kinase-dead and constitutively active PAK4 did not show any significance contribution to the apoptosis property. This may suggest that PAK4 do not operate all kinase domains towards apoptotic function. Immortalized normal ovarian epithelial cell line, HOSE6-3 was also upregulated with PAK4 transfection. However it did not induce the oncogenesis property of cell survival. / published_or_final_version / Pathology / Master / Master of Medical Sciences

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

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