Browsing by Subject "PKC"
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Item Characterization of Protein Kinase C in Cisplatin Sensitive and Resistant Human Cervical Cancer HeLa Cells(2000-12-01) Mohanty, Sanghamitra; Basu, Alakananda; Simecka, Jerry; Dimitrijevich, DanMohanty, S., Characterization of protein kinase C in cisplatin sensitive and resistant human cervical cancer HeLa cells. Master of Science (Microbiology and Immunology), December, 2000. 37 pp., 11 illustrations, bibliography, 27 titles. Signal transduction plays a crucial role in carcinogenesis. A defect in signaling, by evading cell death or promoting cell proliferation, may result in neoplastic transformation or protection of cells from the cytotoxicity of anticancer drugs. Therefore, in order to understand the complex mechanism of drug resistance, it is relevant to probe into the important signal transduction pathways. Protein kinase C, a key signal transducer, influences cisplatin sensitivity in many cell lines. We examined whether or not the PKC signal transduction pathway is affected during development of resistance to cisplatin by tumor cells. PKC activators increased cisplatin sensitivity in both parental and cisplatin-resistant cells. Western blot analysis showed a slight decrease in cPKCα and nPKCε, an evaluation in nPKCδ and no change in the abundance of PKCϚ in HeLa/CP cells compared to HeLa cells. Though TPA-induced translocation of PKC isoforms was identical in both cell lines, down regulation of PKCδ was defective in resistant cells. Therefore, a deregulation in PKCδ was associated with cisplatin resistance.Item Dissecting the Role of Protein Kinase C-Epsilon in Breast Cancer(2013-12-01) Jain, Kirti; Basu, AlakanandaProtein kinase C-epsilon (PKCε) has pro-tumor functions in many cancers including breast cancer. The purpose of this dissertation is to understand the role of PKCε in fundamental processes that are associated with breast cancer development and progression. PKCε is known to promote the survival of breast cancer cells. Autophagy is a process of cellular self-digestion that can mediate cell survival during stress. We have found that PKCε overexpression increases the basal autophagy in breast cancer cells while its depletion reduces it. Moreover, the effect of PKCε on autophagy is isozyme specific. Regulation by PKCε is not limited to basal autophagy as it also mediated starvation-induced autophagy. Looking for the possible mechanisms, we found that PKCε negatively regulates mammalian target of rapamycin (mTOR), which is the master regulator of autophagy. These results show that PKCε positively regulates autophagy, likely, via inhibition of mTOR. PKCε overexpression in mammary epithelial cells led to morphological changes indicating its role in regulation of cell plasticity. Further analysis revealed that PKCε promotes epithelial to mesenchymal transition (EMT), which is an early step in cancer metastasis. In addition, PKCε mediated transforming growth factor-beta (TGFβ)-induced EMT partially via Snail, which is a crucial EMT effector. Moreover, PKCε promoted cell migration and anoikis Ii resistance which are hallmarks of EMT. To examine the phenotypic effect of PKCε manipulation in a physiologically relevant context, we employed three dimensional (3D) cell culture model. We found that PKCε overexpression led to disruption of acinar morphogenesis in 3D culture. These results indicate a causal role for PKCε in breast tumor development and progressionItem Endothelin-1-Induced Signaling Involved in Extracellular Matrix Remodeling(2006-12-01) He, Shaoqing; Thomas Yorio; Neeraj Agarwal; Peter KoulenET-1-Induced Signaling in ECM Remodeling in Astrocytes. Shaoqing He, Department of Pharmacology & Neuroscience, University of North Texas Health Science Center, Fort Worth, TX 76107. ET-1 levels are elevated under pathophysiological conditions, including glaucoma, however, ET-1’s ocular functions are not fully documented. Therefore, ET-1-induced signaling and ECM remodeling in astrocytes and at the optic nerve head were determined in this study. Three signaling pathways, including ERK1/2, PKC, and P13 kinase, were involved in ET-1-medicated cell proliferation of U373MG astrocytoma cells. Blocking one of these pathways completely abolished cell proliferation. It appeared that ERK1/2 activation was involved, but was independent of PKC and P13 kinase activation by ET-1. It was also determined that the ETB receptor was the dominant receptor involved in ERK1/2 phosphorylation and cell proliferation. In addition, ERK1/2 phosphorylation was not transactivated by the EGF receptor by ET-1. The studies also indicated that there was no activation of c/nPKC, although PKC was involved in cell proliferation. In U373MG astrocytoma cells, MAPK-ERK, PKC and P13K pathways appear to exert their roles in parallel without a direct, apparent “cross-talk”. Based on the signaling pathways obtained from U373MG astrocytoma cells, the regulation of MMPs/TIMPs and fibronectin in ET-1-activated human optic nerve head astroctyes (hONAs) was also determined. ET-1 not only induced rapid phosphorylation of ERK1/2 and PKC βI/ βII/δ but also increased the activity of MMP-2 and the expression of TIMP=1 and 2. The activity of MMP-2 was enhanced in the presence of inhibitors of MAPK or PKC in hONAs, whereas the expression of TIMP-1 and 2 was abolished. ET-1 increased the soluble fibronectin (FN) expression as well as FN matrix formation, however, the expression and deposition of FN were MAPK- and PKC-independent, whereas expression and activity of MMps and TIMPs were MAPK- and PKC-dependent. Therefore, ET-1 shifted the balance of MMPs/TIMPs and substrates that altered the ECM composition and subsequently let to ECM remodeling in activated hONA cells. ET-1’s effects on ECM remodeling at the optic nerve head were also examined following intravitreal administration of ET-1 in rats. The increased expression of MMP-9 and collagen VI was detected in both ETB deficient rats and wildtype Wistar rats post ET-1 intravitreal injection for 2 and 14 days, whereas the deposition of FN and collagen IV was unchanged. There was no significant difference in staining of MMP-9 and collagen VI between ETB deficient rats and wildtype Wistar rats. In this study, ECM remodeling was demonstrated in rats injected with ET-1 into the vitreous. Such changes in the ECM seen in the current study provide additional insight into the mechanisms that might explain the glaucomatous changes observed in ET-1-injection or perfusion models. In summary, ET-1 not only activated several signaling pathways in cell proliferation of astrocytes, but also modulated the expression of ECM molecules in vitro and in vivo, indicating that ET-1 plays a regulatory role in ECM remodeling. These effects coupled with observations that ET-1 levels are elevated in glaucoma patients, suggests that ET-1 may be involved in glaucomatous optic neuropathy.Item REGULATION OF PROTEIN KINASE C-ETA IN BREAST CANCER(2013-04-12) Pal, DeepanwitaPurpose: The protein kinase C (PKC) and phosphotidylinositide-3 kinase (PI3K) signaling pathways play critical roles in the development of breast cancer and regulate cell proliferation, differentiation, cell death and tumor promotion. PKCs serve as receptors for tumor-promoting phorbol esters, which are potent activators of conventional and novel members of the PKC family, and can substitute for the physiologic activator diacylglycerol. Prolonged treatment with phorbol esters, however, induces downregulation of these PKCs. Protein kinase C-eta is a novel member of the PKC family but resists downregulation by phorbol esters. This unique regulation of PKC-eta may have implications in tumor promotion. PKCs are regulated not only by cofactors but also by phosphorylation. Phosphorylation and dephosphorylation of PKCs can regulate their activity, stability and function. The objective of this study is to understand the regulation and contribution of PKC-eta in breast cancer. Methods: Established breast cancer cell lines were used in our study. The effect of distinct kinase inhibitors on PKCη protein levels was determined by Western blot analysis. The effect of proteasome and protease inhibitors on PKC-eta levels was also assessed by Western blotting. RNAi technique was utilized to knockdown members of the PKC and PI3K pathways. MTT assay was performed to determine the effect of PKC-eta on cell growth while cell proliferation upon PKC-eta knockdown was monitored by clonogenic assay. Results: Inhibition of PKC and PI3K pathways induced downregulation of PKC-eta via two distinct mechanisms. PKC-eta depletion inhibited the growth and proliferation of breast cancer cells. Conclusions: Our results demonstrate that the distinct regulation of PKC-eta by members of the PKC and PI3K family contributes to breast cancer cell growth.Item Studies of Protein F1 (GAP-43) Expression and Function in Spinal Neuronal Cultures(1994-08-01) El-Badawy, Hassan M.E. Azzazy; Ming-Chi Wu; Guenter W. Gross; Scott NortonEl-Badawy, Hassan M. E. Azzazy, Studies of Protein F1 (GAP-43) Expression and Function in Spinal Neuronal Cultures. Doctor of Philosophy (Biochemistry and Molecular Biology), August 1994, 167 pp., 32 illustrations, References, 194 titles. Protein F1 (GAP-43, B-50, neuromodulin) is a membrane-bound phosphoprotein that has been studied mainly in neurons and is implicated in synaptic plasticity, axonal growth and regeneration, and neurotransmitter release. In this study, a 21 amino acid polypeptide that corresponds to the C-terminus sequence of protein F1 and contains a potential PKC phosphorylation sequence (SXR) was synthesized. The synthetic peptide was phosphorylated by rat PKC in a concentration-dependent manner suggesting that this site in the intact protein may be phosphorylated by PKC in vivo. Polyclonal antibodies against the peptide were produced in a rabbit and used to: (i) recognize native non-phosphorylated protein F1 purified from rat brain, (ii) immunoprecipitate phosphorylated protein F1, and (iii) stain the cell bodies and neuritis of cultured neurons. Electron microscopic studies revealed intracellular protein F1 immunoreactivity but no specific subcellular association of the gold label could be demonstrated. The antibodies were also used to compare protein F1 levels during the development of spinal neurons in culture and in vivo. The highest levels of protein F1 were detected by ELISA, at 2 days in culture. These results are in accordance with previous reports that correlate high expression of protein F1 to neurite outgrowth. In vivo, however, protein F1 reached maximal level at one day after parturition. Two approaches were utilized to investigate the potential physiological functions of protein F1 in spinal neurons networks. First, interaction of positively charged, rhodamine-labeled liposomes with spinal neurons was characterized by fluorescence microscopy and electrophysiological recording. Uniform, non-toxic, and preferential interaction of liposomes with spinal neurons over glia was established. These liposomes were used to deliver anti-protein F1 antibodies into spinal neurons but did not affect neurite formation by these cells. Second, antisense oligodeoxynucleotides internalized into spinal neurons in order to interfere with protein F1 expression had no effect on the development of these cells in culture. Data from this study suggest that Ser-210 at the C-terminus of protein F1 may be a substrate for PKC phosphorylation in vivo. Antibodies raised against F1 peptide revealed protein F1 immunoreactivity that outlined cell bodies and neuritis of cultured spinal neurons. Positively charged liposomes were characterized as a potential delivery system for macromolecules into spinal neurons. Protein F1 levels were shown to be developmentally regulated in mouse spinal neurons in culture and in vivo. Finally, the use of antisense oligodeoxynucleotides against protein F1 mRNA revealed that protein F1 may not be essential for neurite outgrowth of mouse spinal neurons in culture.Item TAAR1-dependent astrocyte dysregulation during HAND and METH exposure(2018-12) Mythen, Shannon; Ghorpade, Anuja; Basu, Alakananda; Barber, Robert C.; Gatch, Michael B.; Krishnamoorthy, Raghu R.Excitatory amino-acid transporter (EAAT)-2 is predominantly expressed in astrocytes and clears glutamate from tripartite synapses preventing excitotoxicity. EAAT- 2 dysregulation occurs during human immunodeficiency virus (HIV)-associated neuroinflammation and methamphetamine (METH) abuse, leading to neurotoxic outcomes. Trace amine associated receptor (TAAR) 1, a METH receptor in astrocytes, triggers EAAT-2 dysfunction. Protein kinase C (PKC) signaling promotes ubiquitination of EAAT-2 C-terminal lysine residues, resulting in EAAT-2 internalization. As a G protein coupled receptor, TAAR1's signaling is implicated in PKC activation. In this work, we investigated the role of TAAR1 in PKC-mediated EAAT-2 ubiquitination during HIV- associated neurocognitive disorders (HAND) and METH comorbidities. We evaluated a TAAR1 overexpression model in primary astrocytes to elucidate TAAR1-mediated functional changes. We found that TAAR1-selective inhibitor, EPPTB, reduced EAAT-2 ubiquitination, and a PKC activator decreased glutamate clearance in METH-pretreated human astrocytes. Therapies targeting astrocyte dysfunction may improve outcomes during HAND, METH abuse and other neuroinflammatory disorders.Item The Regulation of P53 by Protein Kinase C in Anticancer Drug-Induced Apoptosis(2001-12-01) Johnson, Cassie L.; Basu, Alakananda; Wu, Ming-ChiJohnson, C., The regulation of p53 by protein kinase C in anticancer drug-induced apoptosis. Master of Science (Microbiology and Immunology), December, 2001. 43 pp., 11 figures, references, 6 titles. The tumor suppression protein p53 has been implicated in DNA damage-induced apoptosis. Previous studies demonstrated that the protein kinase C (PKC) signal transduction pathway regulates apoptosis induced by the DNA damaging agent cisplatin and is deregulated in cisplatin-resistant cells. The present study examined whether PKC influences p53 and, hence, cellular sensitivity to cisplatin. Basal p53 levels were elevated in cisplatin-resistant HeLa (HeLa/CP) cells as compared to parental HeLa cells. Cisplatin further increased p53 levels in HeLA/CP, but not in HeLA cells. However, rottlerin, a PKC-δ inhibitor that prevents cisplatin-induced apoptosis, caused p53 accumulation in HeLa cells treated with cisplatin. Rottlerin stabilized p53 in response to cisplatin in HeLa cells, whereas cisplatin alone was sufficient to stabilize p53 in HeLa/CP cells.