Date of Award

1-1-2013

Document Type

Open Access Thesis

Department

College of Pharmacy

Sub-Department

Pharmaceutical Science

First Advisor

Igor Roninson

Abstract

The nucleolus is the primary site of ribogenesis and consists of three compartments, namely, fibrillar center (FC), dense fibrillar component (DFC) and granular component (GC). Recent studies have observed a novel structure distinct from these three compartments which forms in the nucleolus upon DNA damage called Intranucleolar body (INoB). Previous studies in our laboratory have found INoBs forming in HT1080 fibrosarcoma cell lines after ectopic overexpression of damage-inducible cell cycle inhibitor p21. This INoB formation has been observed to be decreased by Senexin A which is an inhibitor of transcription regulating kinases CDK8/CDK19.

Here we show that INoB formation correlates with the level of p21 induction in HT1080 fibrosarcoma cells. Similarly, induction of other CDK inhibitors such as p16 and p27 also causes INoB formation which was significantly decreased by Senexin A treatment in case the cells where p16 is overexpressed. DNA damage by doxorubicin also caused the formation of INoBs in HCT116 colon carcinoma cells which was decreased by Senexin A treatment. CDK8 was found to be localized in these INoBs. Comparative studies in HCT116 wt and HCT116 p21-/- cells showed a partial requirement of p21 for DNA damage induced INoB formation and also indicated a possibility of different type of INoBs which we called `type 2 INoBs' which did not contain CDK8 and could arise in the absence of p21

INoB formation was not observed in quiescent cells. Protein content of CDK8, RNA polymerase I and, to a lesser extent, RNA polymerase II was found to be higher in nucleolar extracts of HT1080 cells with p21 overexpression. This indicates that rRNA transcription may be linked to INoB formation and associated changes in the nucleolus. p21 overexpression also caused RNA polymerase I localization at the foci on the periphery of the nucleolus, separate from INoBs, which we called `Pol I aggregates'. Formation of these Pol I aggregates in p21 overexpressed cells was decreased by CDK8 inhibition with Senexin A. INoB formation was not found to be correlated with senescence-associated β-Gal staining (SA-β-Gal), one of the markers of the senescent phenotype, and SA-β-Gal expression was unaffected by Senexin A. However, Senexin A treatment significantly reduced the increase in the cell size in p21-overexpressing HT1080 cells, a characteristic feature of senescence, which is indicative of continued protein synthesis and ribogenesis in senescent cells.

This study reveals additional information regarding the nucleolar restructuring after DNA damage or p21 expression, which may be related to rRNA transcription and cell senescence.

Rights

© 2013, Vineet Garg

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