Abstract
Bioluminescence reporter proteins have been widely used in the development of tools for monitoring biological events in living cells. Currently, some assays like flow cytometry analysis are available for studying DNA synthetic phase (S-phase) targeted anti-cancer drug activity in vitro; however, techniques for imaging of in vivo models remain limited. Cyclin A2 is known to promote S-phase entry in mammals. Its expression levels are low during G1-phase, but they increase at the onset of S-phase. Cyclin A2 is degraded during prometaphase by ubiquitin-dependent, proteasome-mediated proteolysis. In this study, we have developed a cyclin A2-luciferase (CYCA-Luc) fusion protein targeted for ubiquitin-proteasome dependent degradation, and have evaluated its utility in screening S-phase targeted anti-cancer drugs. Similar to endogenous cyclin A2, CYCA-Luc accumulates during S-phase and is degraded during G2/M-phase. Using Cdc20 siRNA we have demonstrated that Cdc20 can mediate CYCA-Luc degradation. Moreover, using noninvasive bioluminescent imaging, we demonstrated accumulation of CYCA-Luc in response to 10-hydroxycamptothecin (HCPT), an S-phase targeted anti-cancer drug, in human tumor cells in vivo and in vitro. Our results indicate that a CYCA-Luc fusion reporter system can be used to monitor S-phase of cell cycle, and evaluate pharmacological activity of anti-cancer drug HCPT in real time in vitro and in vivo, and is likely to provide an important tool for screening such drugs.
Highlights
The DNA synthetic phase (S-phase) of the cell cycle, during which DNA synthesis occurs, plays an important role in cell proliferation [1,2]
Our results indicate that this CYCA-Luc fusion reporter system can be used to monitor pharmacological activity of S-phase-specific anti-cancer drug HCPT in real time in vitro and in vivo, and is likely to provide an important tool for screening such drugs
HCPT caused a dose-dependent increase in luciferase activity in HeLa-CYCALuc cells (Figure 4D). These results demonstrated that HCPT induced S-phase arrest of cell cycle and increased CYCA-Luc expression in cells producing CYCA-Luc under in vitro conditions
Summary
The DNA synthetic phase (S-phase) of the cell cycle, during which DNA synthesis occurs, plays an important role in cell proliferation [1,2]. Many anti-cancer drugs such as DNA topoisomerase I (Topo I) inhibitors target the elongation step of DNA synthesis, and arrest tumor cells in S-phase, subsequently induce apoptosis [3,4]. Parameters in traditional tumor xenograft models such as tumor weight or volume are used predominantly to evaluate drug action These conventional physical measurement techniques require animal sacrifice and prevent real-time monitoring of tumor progression and effects of anti-cancer drugs in living animals. They are not readily adaptable to meet requirements for high-throughput drug screening. New in vivo technologies for screening S-phase-specific anti-cancer drugs are urgently needed
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