Degree Granting Department
Cdk7, Elk-1, ERK1, Oncogene, Signal Transduction
Protein Kinase C-iota (PKC-é), an atypical protein kinase C isoform manifests its potential as an oncogene by targeting various aspects of cancer cells such as growth, invasion and survival. PKC-é confers resistance to drug-induced apoptosis in cancer cells. The acquisition of drug resistance is a major obstacle to good prognosis in neuroblastoma. The focus of the dissertation was three-fold: First to study the role of PKC-é in the proliferation of neuroblastoma. Secondly, to identify the efficacy of [4-(5-amino-4-carbamoylimidazol-1-yl)-2,3-dihydroxycyclopentyl] methyl dihydrogen phosphate (ICA-1) as a novel PKC-é inhibitor in neuroblastoma cell proliferation and apoptosis. Finally, to analyze whether PKC-é could self-regulate its expression. Cyclin dependent kinase 7 (Cdk7) phosphorylates cyclin dependent kinases (cdks) and promotes cell proliferation. Our data shows that PKC-é is an in-vitro Cdk7 kinase and that neuroblastoma cells proliferate via a PKC-é/Cdk7/cdk2 cell signaling pathway. ICA-1 specifically inhibits the activity of PKC-é but not that of PKC-zeta (PKC-æ), the closely related atypical PKC family member. The IC50 for the kinase activity assay was approximately 0.1µM which is 1000 times less than that of aurothiomalate, a known PKC-é inhibitor. The phosphorylation of Cdk7 by PKC-é was potently inhibited by ICA-1. ICA-1 mediates its antiproliferative effects on neuroblastoma cells by inhibiting the PKC-é/Cdk7/cdk2 signaling pathway. ICA-1 (0.1µM) inhibited the in-vitro proliferation of BE(2)-C neuroblastoma cells by 58% (P=0.01). Additionally, ICA-1 also induced apoptosis in neuroblastoma cells. Interestingly, ICA-1 did not affect the proliferation of normal neuronal cells suggesting its potential as chemotherapeutic with low toxicity. Hence, our results emphasize the potential of ICA-1 as a novel PKC-é inhibitor and chemotherapeutic agent for neuroblastoma.
Bcr-Abl has been shown to regulate the activation of the transcription factor ELK-1 which in turn regulates the expression of PKC-é. Alternatively, we hypothesize that PKC-é can self regulate its expression by indirectly regulating the activity of Elk-1 in an ERK1 dependent manner. Our preliminary data shows that there was robust increase in the expression as well as association of PKC-é and Elk-1 in actively proliferating neuroblastoma cells suggesting a potential role of PKC-é in regulating the activity of Elk-1. Analysis of the subcellular fractions also presented a similar increase in the association between PKC-é and Elk-1 in the nuclear fraction of actively proliferating cells as compared to cytoplasm. Interestingly, the nuclear expression of PKC-é was also found to be higher in these cells, suggesting that PKC-é translocated to the nucleus in actively proliferating cells and regulated the transcriptional activity of Elk-1. However, our data from in-vitro kinase activity demonstrated that PKC-é was not an Elk-1 kinase but that it increased the phosphorylation of Elk-1 in the presence of ERK1, an upstream kinase of Elk-1 in the Bcr-Abl mediated regulatory pathway of PKC-é. This suggested that ERK1 was integral to the self-regulatory activity of PKC-é. In conclusion, we hypothesize that the self-regulatory mechanism of PKC-é is initiated by the translocation PKC-é into the nucleus where it activates ERK1. This promotes the activation of its downstream target Elk-1 which subsequently upregulates the expression of PKC-é
Scholar Commons Citation
Pillai, Prajit P., "Role of Protein Kinase C-iota in Neuroblastoma and the Effect of ICA-1, a Novel Protein Kinase C-iota Inhibitor on the Proliferation and Apoptosis of Neuroblastoma Cells" (2011). Graduate Theses and Dissertations.