Primary response genes (PRGs) are a group of genes that are rapidly induced when cells - PowerPoint Presentation

1. Primary response genes (PRGs) are a group of genes that are rapidly induced when cells experience an abrupt change in their environment. The most common abrupt changes in cellular environment are typically acute injuries, but can also include exposure to a chemical or other substance. PRG activation is controlled by a single kinase, CDK9 (5). BACKGROUNDSample #Morphine presentCdk9 inhibitor(flavopiridol)Time (h)M1 (control)NoNo3M2YesNo1,3M3YesYes1,3M4NoYes1,3PRG’S AND OPIOID TOLERANCE/DEPENDENCEBIG PICTURE1. Identify a cell lineage with a mu opioid receptor. 2. Treat the cells under the following conditions:3. Isolate RNA from these cells.4. Perform qRT-PCR with a few known response genes to verify RNA quality and appropriate cellular responses. 5. Submit samples for RNA sequencing. 6. Analyze RNA sequencing data to determine if opioid PRG’s are induced by Cdk9-dependent pathways and if these pathways are inhibited by the Cdk9 inhibitor.EXPERIMENTAL DESIGNNEXT STEPS1. Al-Hasani, R., Bruchas, M. Molecular Mechanisms of Opioid Receptor-Dependent Signaling and Behavior. Anesthesiology. 2011 December. 2. What is the U.S. Opioid Epidemic? Department of Health and Human Services. Sep 4 2019. https://www.hhs.gov/opioids/about-the-epidemic/index.html3. J.C. Ballyntyne, J. Mao Opioid therapy for chronic pain. New Engl J Med 349 2003 1943–1953.4. J. Cami, M. Farré Drug addiction. New Engl J Med 349 2003 975–986.5. Yik, J.H.N., Chen, R., Nishimura, R., Jennings, J., Link, A.J., and Zhou, Q. Inhibition of P-TEFb (CDK9/cyclin T) kinase and RNA polymerase II transcription by the coordinated actions of HEXIM1 and 7SK snRNA. Mol. Cell (2003)12: 971-982REFERENCESAn inhibitor of opiate tolerance and dependence pathways could have a very significant and positive impact on the current opiate crisis and could be a potentially useful tool in battling opiate addiction. Dominik Haudenschild PhD, Jasper Yik PhD, Sarah Williams UC Davis Department of Orthopedic Surgery The effect of a cdk9 inhibitor on opioid primary response genes, tolerance, and dependence Continue to identify appropriate cell lineages and optimize experimental conditions to get appropriate cell responses. Run experiment, analyze RNA sequencing data. THE OPIOID CRISISThe Department of Health and Human Services declared the Opioid Crisis a public health emergency in 2017. According to HHS numbers, 2.0 million people had an opioid use disorder in 2018 and about 130 people died every day from overdosing on opioids (2). Use of opioids is known to induce tolerance and physical dependence (3,4).Exact mechanisms of opioid tolerance signaling are still relatively unknown and are somewhat controversial (1). Some theories include: Downregulation of opioid receptors (including KOR, MOR, DOR, ORL-1) Receptor desensitization and uncoupling from downstream signaling pathways Ineffective regulation of cAMP by opiates that leads to an increase in cAMP and an increase in tolerance Agonist-induced receptor phosphorylationHow cells change gene expression levels when they are first exposed to opiates is still unknown and might be useful in better understanding the opiate response. Knowing this response could help us better understand tolerance mechanisms of opiates and we might, therefore, be able to reduce the addictiveness of opiates. OBJECTIVEOur objective is to determine whether we can identify Cdk9-dependent PRGs that are activated when cells are exposed to opiates. In doing so, we could answer these questions: Will transcription of genes induced by an opiate be blocked by the Cdk9 inhibitor? Will the Cdk9 inhibitor inhibit genes implicated in opiate tolerance and dependence pathways?Acute Injury orExposure to chemical/ other substance Multiple signaling pathways CDK9Single rate-limiting bottleneck Multiple cellular pathwaysTolerance?Dependence?CDK9 Inhibitor INTERIM RESULTSThe experiment was conducted using human neuroblastoma cells and morphine. Using qRT-PCR, we looked for RNA products of these genes: Elk1, Myc, Jun, Fos, Srf, HoxB9.No significant RNA products were identified for any of the targeted genes.