Date of Award
Bachelor of Science
apoptosis, cell, protein, intracellular
Apoptosis is a highly organized intracellular death program in multicellular animals. In the vertebrate immune system, apoptosis plays a central role in preventing the emergence of autoreactive lymphocytes. In this study, we used multiple stimuli (staurosporine, camptothecin, and cytokine deprivation - interleukin-2 or IL-2) to initiate apoptosis in IL-2 dependent, mouse HT-2 T-lymphocytes. All three inducers triggered DNA laddering and phosphatidylserine externalization. Propidium iodide staining and flow cytometry were also used to determine whether apoptotic cells accumulated in a specific stage of the cell cycle, and whether the mode of induction affected cell cycle distribution. Our findings indicate that IL-2 deprivation induces HT-2 cells to accumulate in G1, while cells treated with staurosporine and camptothecin accumulated in G2/M and S phase, respectively. We also used immunopblotting detection to investigate P27Kip1 protein expression for each condition. P27Kip1 is a member of the Cip/Kip family of cyclin-dependent kinase inhibitors whose function is to enforce the G1 restriction point. We found that P27Kip1 levels were significantly increased within 12 hrs and 6 hrs following both IL-2 deprivation and campthotecin exposure, respectively. Across all time points in the staurosporine experiments, P27Kip1 steady state levels were decreased. These results suggest that the mode by which apoptosis is induced differentially affects cell cycle distribution.
Pinkston, Nell, "Flow Cytometry and Biochemical Analysis of Apoptotic Mouse HT-2 T - Lymphocytes" (2015). Honors Theses and Student Projects. 374.