Hierarchy of a regenerative cell cycle: Cyclin E1 multitasks
Authors: Sabrina A. Stratton, Michelle Craig Barton
Journal: Hepatology (2013)

Disruptions in mechanisms that control cell cycle occur frequently in human cancers with loss or derangement of function by specific controllers of cell cycle. This in turn leads to increased proliferation and subversion of checkpoints, which normally keep genomic and chromosomal instability at bay.1 To apply this knowledge in development of targeted therapeutic treatments demands a deep understanding of how the cell cycle is controlled in normal cells, especially given the complexities of mammalian systems where key regulatory players may have multiple isoforms, cryptic functions, and apparent redundancy. Each phase of the cell cycle is characterized by specific drivers: protein complexes of cyclins and their associated kinases (cyclin-dependent kinase [Cdk]) that generate waves of phosphorylation signals, initiated by the availability of specific cyclins to activate partner kinases, and propel cell cycle forward. In mammals, the classical view is there are interphase Cdk's, Cdk2, 4, and 6, and mitotic Cdk1, along with 10 cyclin proteins, divided among A, B, D, and E classes. Cdk4 and 6 are activated by binding to mitotic D-type cyclins during G1. These complexes are involved in phosphorylation and inactivation of retinoblastoma (RB) proteins, which allows expression of cyclin E proteins. Cdk2 in association with cyclin E further inactivates RB proteins, to facilitate expression of cyclin A proteins that in turn associate with Cdk2 to promote progress through S-phase into G2 and mitosis.1-4 Studies with genetically modified mouse models unveiled additional complexities to this paradigm: Cdk1 may be the only essential Cdk, as Cdk1−/− mice fail to undergo cell division during development, and Cdk1 is sufficient for this process. However, this is not true in all cell types: conditional loss of Cdk1 in hepatocytes shows that Cdk1 is not essential for DNA replication in these cells and these mice are able to regenerate liver tissue in response to surgical resection.5, 6 Partial, surgical resection of the liver (partial hepatectomy [PH]) forces normally quiescent cells to undergo a robust and relatively synchronized process of cell cycle entry and progression until liver mass is restored. The regenerative cell cycle is induced in response to surgery with limited necrosis and inflammation.8 Thus, this system offers unique access to understanding how nontransformed, postmitotic cells reenter and undergo cell cycle. However, whether mechanisms defined in PH-induced regeneration are applicable in liver disease, amid chronic inflammation, or in other regenerative paradigms with different cell types must be established empirically. In this issue of Hepatology, Hu et al.7 use a systematic, multifaceted approach of complex genetics and surgically induced regeneration of liver tissue in mouse models, alongside in vitro assessments of mechanism in primary hepatocytes, to define how complexes of Cdk2 and E-type cyclins (CcnE1 or CcnE2) regulate cell cycle in regenerative hepatocytes. The current study shows how CcnE1 assumes critical kinase-independent functions, in the absence of CcnE2, and drives prereplication complex loading, Cyclin A2 expression and S-phase progression. CcnE2 is not equally competent: Cdk2 is essential for CcnE2 function, in the absence of CcnE1, and expansion of a progenitor Cdk2+ population occurs to meet the demands of regeneration when hepatocytes lack CcnE1 and Cdk2. Mammalian cyclin E (CcnE) has two distinct isoforms, CcnE1 and CcnE2, which are expressed during G1/S transition and degraded during S phase in a Cdk2-dependent manner. Previous attempts to untangle the relationship of CcnE2 and CcnE1 with Cdk2 by single gene deletions showed that CcnE1−/− mice display a relatively normal regenerative response with a minor delay in S-phase entry. In contrast, CcnE2−/− mice overexpress CcnE1 and "hyperactivate" Cdk2, resulting in accelerated and extended DNA synthesis, hepatomegaly, and rampant endoreduplication in response to PH.9 These findings compelled the authors of the current study to generate mouse models with combinations of CcnE1, CcnE2 and hepatocyte-specific Cdk2 gene deletions.7 Cell cycle entry remains normal in regenerating Cdk2Δhepa liver, as increased Cdk1 kinase activity in Cdk2Δhepa hepatocytes likely compensates, confirming Cdk2 dispensability.10 However, the combined loss of Cdk2 and CcnE1 in vivo inhibits S-phase progression. Likewise, with complete loss of cyclin E, CcnE1−/−;CcnE2−/− mice have impaired liver regeneration, similar to Cdk2Δhepa;CcnE1−/− mice. The loss of Cdk2, in triple knockout Cdk2Δhepa;CcnE1−/−;CcnE2−/− mice, causes no additional deficiencies, supporting Cyclin E as key.7 As cell cycle progresses further into S-phase, the expression levels of cyclin A (isoforms CcnA1 and CcnA2) increase and CcnA associates with Cdk2 to facilitate DNA synthesis.11-13 In the current study, when Cdk2 is not available to associate with CcnA in Cdk2Δhepa;CcnE1−/− hepatocytes,7 there is diminished Cdk1 and CcnA2 activation in vitro. CcnE1 may also activate CcnA2 expression, as CcnE1 is recruited to the CcnA2 promoter in cultured primary hepatocytes. Without CcnE1 functions in Cdk2Δhepa;CcnE1−/− mice, there is loss of S-phase progression and impaired liver regeneration, unlike Cdk2Δhepa;CcnE2−/− mice. Cyclin E also functions in late M/early G1 phase to preload MCM2-7 helicase complexes, recruited by Cdc6 and Cdt1, at origins of replication and initiate one round of DNA synthesis per cell cycle.14, 15 In the current study, analysis of MCM2 retention in chromatin, fractionated from Cdk2Δhepa;CcnE1−/−, Cdk2Δhepa and control primary hepatocytes, suggests it is unphosphorylated CcnE1 that performs the loading of MCM proteins onto chromatin. Liver regeneration is a process that involves the whole organ, as emphasized by comparison of isolated, primary Cdk2Δhepa;CcnE1−/− hepatocytes to post-PH liver of Cdk2Δhepa;CcnE1−/− mice. Hu et al. found that the drive to regenerate Cdk2Δhepa;CcnE1−/− liver induces expansion of a Cdk2+ population of nonparenchymal progenitor cells that support restoration of liver mass. Lacking this fail-safe ability to exploit a progenitor niche, primary hepatocytes without CcnE1 and Cdk2 have high levels of cell death in response to mitogens. The mechanisms whereby genetic inhibition of the S-phase machinery in the liver triggers expansion of a progenitor cell population are not completely understood. Liver function in Cdk2Δhepa;CcnE1−/− mice is preserved by both progenitor cell activation and compensatory hepatocyte hypertrophy. The authors recently reported that CcnE1, not CcnE2, was essential for hepatic stellate cell proliferation and survival.16 In the current study, the authors stop short of identifying these cell populations in their models and their contribution to liver regeneration. Perhaps future work will provide more insight into these questions, and further determine whether the multitasking of Cyclin E1 occurs in hepatocytes exposed to chronic inflammation and injury of liver disease or other regenerative cell systems. Sabrina A. Stratton, B.S.1 Michelle Craig Barton, P.h.D.1,2 1Department of Biochemistry and Molecular Biology, Center for Stem Cell and Developmental Biology, UT MD Anderson Cancer Center, Houston, TX 2Graduate Program in Genes and Development, University of Texas Graduate School of Biomedical Sciences, Houston, TX