Poster abstracts

Poster number 44 submitted by John Sherman

A metabolic checkpoint in G2

John Sherman (MCDB, Nationwide Childrens hospital), Yuqing Shen (Nationwide Childrens hospital), Ruoning Wang (Nationwide Childrens Hospital)

Abstract:
Significance
Integration of cell cycle regulation and metabolic status is a critical yet poorly defined aspect of malignancy. A key factor in malignancy is the abrogation of the more robust G1 checkpoint to achieve uncontrolled proliferation. This leaves the G2 checkpoint to protect against lethal mutations or metabolic stresses. Meanwhile, cancer cells alteration of their metabolic flux in response to extracellular nutrients is necessary to thrive in the range of nutrient restrictive environments found throughout the tumor. We use the term “metabolic checkpoint” to describe the signaling response that permits a coordinated adaptation of cell cycle progression as well as metabolic flux in response to extracellular nutrients. In this study we aim to identify key regulatory steps in the G2 metabolic checkpoint. By preventing metabolic checkpoint activation we hope to reduce the fitness of cancer cells under typical stresses experienced in the tumor.
Methods
By synchronizing cells at the G1/S phase boundary of the cell cycle, using a double thymidine block, we can test the effects of chemical insult against G2 specific regulatory mechanisms. These cells can then be separated into distinct cell cycle phases using flow cytometry. Cells subjected to siRNA knockdown are incompatible with double thymidine block, and are instead assayed using a DNA synthesis measuring approach to separate G2 phase cells from an unsynchronized population during analysis. Cellular metabolism is assayed by measuring the release of radioactive tracers from labeled metabolites.
Results
This checkpoint is not mediated by ATP or NAD+, but can be bypassed through chemical or genetic manipulation of cell cycle regulator Wee1 or MEK1/2 of the MAPK signaling cascade. Activation of the checkpoint confers increased fitness during metabolic stress by enabling the metabolism of alternative nutrients and preserving viability.

Keywords: Metabolism, Cell Cycle