Bioluminescent imaging of Cdk2 inhibition in vivo

Abstract

Many proteins and pathways of pharmaceutical interest impinge on ubiquitin ligases or their substrates. The cyclin-dependent kinase (Cdk) inhibitor p27, for example, is polyubiquitylated in a cell cycle-dependent manner by a ubiquitin ligase complex containing the F-box protein Skp2. Regulated turnover of p27 is due, at least partly, to its phosphorylation by Cdk2 on threonine 187, which generates a Skp2-binding site. We made a p27-luciferase (p27Luc) fusion protein and show here that its abundance, like that of p27, is regulated by Skp2 in a cell cycle-dependent manner. As predicted, p27Luc levels increased after blocking Cdk2 activity with inhibitory proteins, peptides or small interfering RNA (siRNA). Accumulation of p27Luc in response to Cdk2 inhibitory drugs (flavopiridol and R-roscovitine) was demonstrable in human tumor cells in vivo using noninvasive bioluminescent imaging. In theory, the approach described here could be used to develop bioluminescent reporters for any drug target that directly or indirectly affects the turnover of a ubiquitin ligase substrate.

Visualizing the dynamics of p21 Waf1/Cip1 cyclin-dependent kinase inhibitor expression in living animals

“…For the detection of luciferase expression, mice were anesthetized, injected i.p. with D-luciferin sodium salt (75 mg/kg) 5 min before beginning photon recording (16)(17)(18)(19)(20). Mice were placed in the light-tight chamber, and a gray-scale image of the mice was first recorded with dimmed light, followed by acquisition of luminescence image by using a cooled CCD camera (Princeton Instruments, Trenton, NJ).…”

“…Optical imaging by bioluminescence allows a noninvasive and real-time analysis of various biological responses, such as gene expression, proteolytic processing, or protein-protein interactions, in living animals (16)(17)(18)(19)(20). In this study, we generated a transgenic mice line (p21-p-luc) expressing the firefly luciferase under the control of the p21 Waf1 driven by the p21 Waf1/Cip1 gene promoter, which contains two p53-binding sites, was established and subjected to noninvasive in vivo BLI.…”

Romo1 is a negative-feedback regulator of Myc

“…The other siRNA against Romo1 was described previously (Hwang et al, 2007). The Myc siRNA sequences (Grandori et al, 2005), Skp2 siRNA sequences (Carrano et al, 1999;Nishitani et al, 2006;Zhang et al, 2004), and control siRNA sequence (Chung et al, 2009) were described previously. siRNAs were purchased from Bioneer (Taejon, Republic of Korea).…”

Repair, Abort, Ignore? Strategies for Dealing With UV Damage

“…Presence of unrepaired DnA damage is a powerful trigger for halting the cell cycle until damage is repaired or, if it is deemed irreparable -for activating the cellular program for apoptosis. Generally, in order to avoid replication of damaged DnA, its structural and sequence integrity is assessed in several checkpoints, each governing a transition point in the cell cycle, and damage is repaired before the cell cycle could proceed further (13,37,48). it has been proposed that for some cells that are not expected to divide further, such as terminally differentiated cells, damage assessment and repair is limited to actively transcribed genes only (7,8,9,31).…”