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The G1 phase optical reporter serves as a sensor of CDK4/6 inhibition in vivo.

ABSTRACT: Visualization of cell-cycle G1 phase for monitoring the early response of cell cycle specific drug remains challenging. In this study, we developed genetically engineered bioluminescent reporters by fusing full-length cyclin E to the C-terminal luciferase (named as CycE-Luc and CycE-Luc2). Next, HeLa cell line or an ER-positive breast cancer cell line MCF-7 was transfected with these reporters. In cellular assays, the bioluminescent signal of CycE-Luc and CycE-Luc2 was accumulated in the G1 phase and decreased after exiting from the G1 phase. The expression of CycE-Luc and CycE-Luc2 fusion protein was regulated in a cell cycle-dependent manner, which was mediated by proteasome ubiquitination and degradation. Next, our in vitro and in vivo experiment confirmed that the cell cycle arrested by anti-cancer agents (palbociclib or 5-FU) was monitored quantitatively and dynamically by bioluminescent imaging of these reporters in a real-time and non-invasive manner. Thus, these optical reporters could reflect the G1 phase alternation of cell cycle, and might become a future clinically translatable approach for predicting and monitoring response to palbociclib in patients with ER-positive breast cancer.

SUBMITTER: Guo C

PROVIDER: S-EPMC7975702 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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The G1 phase optical reporter serves as a sensor of CDK4/6 inhibition <i>in vivo</i>.

Guo Cuiping C Guo Yuxian Y Liu Jingjing J Gao Yiyang Y Wei Min M Zhao Ruijun R Chen Min M Zhang Guojun G

International journal of biological sciences 20210131 3

Visualization of cell-cycle G1 phase for monitoring the early response of cell cycle specific drug remains challenging. In this study, we developed genetically engineered bioluminescent reporters by fusing full-length cyclin E to the C-terminal luciferase (named as CycE-Luc and CycE-Luc2). Next, HeLa cell line or an ER-positive breast cancer cell line MCF-7 was transfected with these reporters. In cellular assays, the bioluminescent signal of CycE-Luc and CycE-Luc2 was accumulated in the G1 phas ...[more]

PMID: 33767584

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Project description:The retinoblastoma tumor suppressor protein (Rb) regulates early G1 phase checkpoints, including the DNA damage response, as well as cell cycle exit and differentiation. The widely accepted model of G1 cell cycle progression proposes that cyclin D:Cdk4/6 partially inactivates the Rb tumor suppressor during early G1 phase by progressive multi-phosphorylation, termed hypo-phosphorylation, resulting in release of E2F transcription factors. However, this model remains largely unproven biochemically and the biologically active form(s) of Rb remains unknown. Here we find that Rb is un-phosphorylated in G0 cells and becomes exclusively mono-phosphorylated throughout all of early G1 phase by cyclin D:Cdk4/6. Early G1 phase mono-phosphorylated Rb is composed of 14 independent isoforms that are all targeted by the E1a oncoprotein, but each shows a preferential binding pattern to specific E2F1-4 transcription factors. At the late G1 Restriction Point, cyclin E:Cdk2 inactivates Rb by a quantum hyper-phosphorylation (>12 phosphates/Rb). Cells undergoing a DNA damage response activate cyclin D:Cdk4/6 to generate mono-phosphorylated Rb that regulates global transcription. In contrast, a non-phosphorylatable ?Cdk-Rb allele was non-functional for regulating a DNA damage response, but functional for driving cell cycle exit and differentiation during myogenesis. These observations fundamentally change our understanding of G1 cell cycle progression and show that there is no progressive multi-phosphorylation or hypo-phosphorylation inactivation of Rb during early G1 phase by cyclin D:Cdk4/6. Instead, cyclin D:Cdk4/6 generates functionally active, mono-phosphorylated Rb that is the only Rb isoform present in cells during early G1 phase. Global transcriptional analysis of murine embryonic fibroblasts (MEFs) with conditional deletion of the endogenous RB gene by treatment with cell permeable TAT-Cre. Comparison to unaltered MEFs and MEFs with physiological level of exogenous wildtype or phospho-mutant RB expressed at time of RB gene deletion.

Dataset Information

The G1 phase optical reporter serves as a sensor of CDK4/6 inhibition in vivo.

Publications

The G1 phase optical reporter serves as a sensor of CDK4/6 inhibition <i>in vivo</i>.

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