Date of Award
My dissertation includes two projects. The first project is focused on the regulation of glycolysis and oxidative metabolism by inducing heme synthesis in cancer cells. The second project deals with the effect of hydroxyurea on RNA synthesis. Heme is an essential cofactor for enzymes of the electron transport chain (ETC) and ATP synthesis in mitochondrial oxidative phosphorylation (OXPHOS). Heme also binds to and destabilizes Bach1, a transcription regulator that controls expression of several groups of genes important for glycolysis, ETC, and metastasis of cancer cells. Since many cancers are characterized by a high glycolytic rate regardless of oxygen availability, targeting glycolysis, ETC, and OXPHOS have emerged as a potential therapeutic strategy. Here, we report that enhancing heme synthesis through exogenous supplementation of heme precursor 5-aminolevulinic acid (ALA) suppresses oxidative metabolism as well as glycolysis and significantly reduces proliferation of both ovarian and breast cancer cells. ALA supplementation also destabilizes Bach1 and inhibits migration of both cell types. Promoting heme synthesis by ALA supplementation may thus represent a promising new anti-cancer strategy, particularly in cancers that are sensitive to altered redox signaling. In the second project, we investigated transcription changes triggered by DNA damage response (DDR). These changes depend on the on the nature of DNA damage, activation of checkpoint kinases, and stage of cell cycle. The transcription changes can be localized and affect only damaged DNA, but they can also be global and affect genes that are not damaged. While the purpose of localized transcription inhibition is to avoid transcription of damaged genes and to make DNA accessible for repair, the purpose and mechanisms of global transcription inhibition of not damaged genes is less well understood. We show here that a brief cell treatment with hydroxyurea (HU) globally inhibits RNA synthesis and transcription by RNA polymerase I, II, and III (RNAPI, RNAPII, and RNAPIII). HU triggers RNAPII accumulation at the 3’ ends of genes, indicating defect in transcriptional termination and pre-mRNA 3’ end processing. Interestingly, we observe destabilization of mRNAs after HU treatment. Experiments with cells defective in individual stages of mRNA decay pathway suggest that mRNAs produced in the presence of HU contain shorter poly(A) tails that do not require deadenylation for subsequent degradation, or that a decay step subsequent to deadenylation is activated by HU. The HU-triggered RNAPII accumulation at the 3’ ends of genes is exacerbated when the checkpoint kinase Mec1p is inactivated, suggesting involvement of DNA damage checkpoint in transcriptional termination
Kaur, Pritpal, "HYDROXYUREA GLOBALLY INHIBITS RNA SYNTHESIS, DESTABILIZES mRNAs, AND TRIGGERS MEC1-OPPOSED RNAPII ACCUMULATION AT 3’ ENDS OF GENES" (2023). Theses and Dissertations. 522.