Cyclin E1, the prototypic E-cyclin, was first described in 1991 1 , and has since been found to have crucial roles in cell proliferation and oncogenesis 2 3 . The second mammalian E-cyclin, cyclin E2, was identified in 1998 4 5 , and is largely regarded as being functionally redundant with cyclin E1 2 3 6 . Cyclin E1 and cyclin E2 are encoded by different genes: cyclin E1 by CCNE1 at 19q12, and cyclin E2 by CCNE2 at 8q22.1. The cyclin E1 and cyclin E2 proteins display high sequence similarity (69.3% in Homo sapiens), and important functional motifs are conserved. These include domains for Cdk (cyclin dependent kinase) and Cdk inhibitor interaction, a nuclear localisation sequence and a centrosome localisation sequence (Figure 1). This high sequence conservation has supported a hypothesis of complete redundancy between the two proteins.

More recent data have identified instances of specific regulation or function for each E-cyclin. First, animal models hint that we have not fully delineated the roles of the E-cyclins, and cyclin E2^-/- mice display subtle phenotypes that may indicate key functional differences to cyclin E1. A second difference is that cyclins E1 and E2 can be regulated by distinct transcription factors and miRNAs. Finally, the expression of cyclin E1 and E2 is not always linked in cancer, and this discordance confirms that there are likely to be underlying functional and regulatory differences between the two proteins.

The E-type cyclins activate the kinase Cdk2 that phosphorylates substrates including the retinoblastoma protein (Rb). Rb phosphorylation leads to the release of E2F transcription factors and initiation of S phase and DNA synthesis, by induction of expression of S phase proteins including histone proteins and cyclin A. Cyclin E-Cdk2 also directly phosphorylates proteins involved in centrosome duplication (NPM, CP110, Mps1), DNA synthesis (Cdt1), DNA repair (Brca1, Ku70), histone gene transcription (p220/NPAT, CBP/p300, HIRA) and Cdk inhibitors p21^Waf1/Cip1 or p27^Kip1 (reviewed in 2 3 7 ). The specificity of cyclin-Cdk activity towards particular substrates is predominantly mediated via differences in cyclin sequence and periodic expression of cyclins during cell cycle phases, along with specific sub-cellular localisation 8 9 . Given that cyclins E1 and E2 are very similar in sequence and are both nuclear proteins 5 10 , it seems probable that cyclin E1-Cdk2 and cyclin E2-Cdk2 phosphorylate a very similar subset of proteins so long as they exhibit the same periodicity of expression. There is considerable overlap even between cyclin E1-Cdk2 and cyclin A-Cdk2 targets 8 . Cyclin E1 can also activate Cdc2/Cdk1. In Cdc2 knockout mice, cyclin E1-Cdc2 kinase activity compensates for the absence of cyclin E1-Cdc2 activity to promote S phase entry 11 . Cyclin E1 also interacts with Cdc2 in the presence of Cdk2, which possibly contributes to S-phase entry in mitotic cell cycles 11 . Both E-cyclins can also complex with Cdk3, although it is not known if this interaction is significant in vivo 5 12 .

While a predominant function of the E-cyclins is to activate Cdk2, it has become apparent that there are crucial Cdk-independent roles (Figure 2). Cdk2^-/- mice are viable whereas cyclin E1^-/- E2^-/- mice are embryonic lethal 13 , implying an essential Cdk-independent function for the E-cyclins. Furthermore, truncated variants of cyclin E1 that cannot bind Cdk2 are able to induce malignant transformation 12 , and oncogenesis has been associated with increased cyclin E1 in the absence of increased Cdk2 activity 12 14 15 16 . Subsequent to these studies, additional major roles for cyclin E1 have been established in the formation of pre-replication complexes on DNA, endocycling and centrosome duplication (Figure 2).

In order for DNA synthesis to occur after the transition from quiescence (G₀) into the cell cycle, it is necessary for the DNA replication complexes to be assembled de novo at the origins of replication. These pre-initiation replication complexes (pre-RC) consist of the pre-licensing factors Cdt1 and Cdc6 that associate with the origin-binding protein ORC, and the DNA replicative helicase components, MCM 2-7 17 . Cyclin E1, independent of Cdk2 activity, associates with DNA near replication origins, and facilitates MCM loading at origins through direct interactions with MCM proteins and Cdt1 18 . This process may also be important in endoreplication, where DNA is replicated without cell division. Endoreplication results in polyploid cells that have essential functions in development, cell differentiation and as an energy reserve 19 . E-cyclins are crucial for endocycling, with the absence of E-cyclins leading to reduced DNA copy number and mortality due to failures in the polyploid giant trophoblast cells of the placenta 13 20 . Like diploid cells, endocycling cells require pre-RC assembly at origins of replication. While the precise function of the E-cyclins in endocycling is not established, in Drosophila cyclin E recruits MCM2 to the DNA during early endocycles of polytene cells of the salivary gland 21 , implying that E-cyclins also function in pre-RC formation in these cells.

A 20 amino acid centrosome localisation sequence (CLS) in cyclin E1 targets this protein to the centrosome 22 . Cyclin E1 overexpression increases the proportion of cells in S phase by a mechanism that is dependent upon this region, but independent of Cdk2-binding 22 . The increase in the proportion of S phase cells may reflect a lengthening of S phase, rather than an increase in proliferation per se 23 . In fact, the cyclin E1 CLS is responsible for co-localising MCM5 to the centrosome, where its presence inhibits centrosome over-duplication and proliferation 24 . Consequently the CLS may function as a coordinating link between centrosome duplication and pre-RC formation 24 . Cdk2 activity is also synchronised with these events, as cyclin E1-Cdk2 phosphorylates nucleophosmin, CP110 and Mps1, promoting centrosome duplication 2 3 7 .

The non-Cdk functions of the E-cyclins have been investigated using cyclin E1, and have been presumed to be identical for cyclin E2 18 22 . The CLS motif is well conserved between cyclin E1 and cyclin E2, implying that cyclin E2 would be functionally active at the centrosome 18 22 . Cyclin E1^-/- E2^-/- mice are embryonic lethal whereas the individual knockout mice have largely normal phenotypes, which supports an assumption that either cyclin E1 or E2 can fulfil all of the functions of the E-cyclins. Despite this apparent redundancy, a careful consideration of animal models leads us to question whether cyclin E1 and cyclin E2 are true homologs.

The E-cyclins are cell cycle regulated at both the mRNA and protein level, leading to cell cycle phase-specific expression. Cyclin E1 and E2 mRNA (CCNE1 and CCNE2) peak in mid-G₁ to early S phase in multiple models of mitotic division 4 5 10 . During S phase cyclin E1 is rapidly downregulated via proteosomal degradation (for reviews of cyclin E1 proteosomal degradation see 2 49 ). In brief, during early S phase cyclin E1 is phosphorylated at conserved residues via Cdk2 and GSK-3β, leading to recognition and ubiquitination of cyclin E1 by the ubiquitin ligase SCF^Fbw7, followed by degradation during S phase 2 49 . The turnover of cyclin E2 is reported to be regulated in a similar manner 50 but has been examined in less detail. Cyclin E1 turnover can also be mediated by ubiquitin ligase components Skp2 51 , Parkin 52 and Cul4 53 , which may contribute to late S-phase degradation of cyclin E1. The combination of transcriptional and post-translational regulation results in an intense peak in expression of cyclin E1 in late G₁ and early S phase of the cell cycle. The activity of cyclin E1-Cdk2 and cyclin E2-Cdk2 complexes is further refined through binding of the Cdk inhibitors p21^Waf1/Cip1 and p27^Kip1, whose expression is also cell cycle regulated.

The cell cycle dependent transcription of the E-cyclins is mediated by E2F transcription factors, which are activated via release from Rb during late G₁ phase. Rb-deficient cells have high expression of cyclin E1 and cyclin E2 45 54 , likely due to constitutive E2F release, and numerous gene expression array studies have confirmed both CCNE1 and CCNE2 as E2F1, E2F2 and E2F3 target genes 55 56 57 . E2F proteins interact with multiple co-regulators at the E-cyclin promoters, allowing for the input of mitogenic signals. E2F1 recruits the histone acetylase p300/CBP 58 and the co-activator SRC3 59 to the CCNE1 promoter. This complex further recruits the protein methyltransferase Carm1/PRMT4 to the CCNE1 and CCNE2 promoters 60 , leading to increased transcription of at least the CCNE1 gene 60 61 . CCNE1 transcription is actively inhibited by Rb and the other pocket proteins in G₀ and G₁ arrested cells 58 60 62 63 64 65 66 and during late mitosis 67 . Rb, via an interaction with inhibitory E2Fs (E2F4-6) recruits the histone deacetylase HDAC1 58 62 63 , the methylation complex SUV39H1 and HP1 64 65 and the BRG1/hBRM nucleosome remodeling complex 66 to the CCNE1 promoter, leading to inhibitory deacetylation, methylation and remodelling of the promoter-associated nucleosomes. The methlytransferase PRMT5, via binding partner COPR5 68 69 , also negatively regulates CCNE1 and CCNE2 transcription 70 71 , especially during G₀ 60 .

Cyclin E1 is a well established oncogene, and its overexpression, especially in a hyperstable form, leads to increased incidence of mouse neoplasia 94 95 96 , and increased susceptibility to other oncogenes 96 . Potential oncogenic effects of cyclin E2 have not been examined in mice, except that mouse embryonic fibroblasts from the E-cyclin double knockout mice are not susceptible to transformation 13 . Further evidence from cell culture models indicates that cyclin E2 has similar proliferative effects to cyclin E1 in cancer cells. The overexpression of either cyclin E1 or cyclin E2 leads to a redistribution of cells within cell cycle phases, with a shorter G₁ 5 , and a longer S phase at least in cyclin E1 overexpressing cells 23 31 . The siRNA-mediated decrease of either E-cyclin severely attenuates the estrogen-induced proliferation of breast cancer cells 75 , and the reduction of either cyclin E1 or E2 leads to reduced colony forming ability in oral squamous cell carcinoma cells, although cyclin E1 ablation is more potent than ablation of cyclin E2 (70% vs 20% reduction) 97 . Cyclin E1 may have higher potency than cyclin E2 as it can be cleaved into low molecular weight fragments with enhanced oncogenic activity 98 , and cyclin E2 does not appear to be similarly processed 99 .

While cyclin E1-Cdk2 and cyclin E2-Cdk2 kinase activities are increased in breast cancer compared to normal tissue 83 , cyclin E1 and E2 expression and kinase activity are not essential for proliferation of all cancer cell types 97 . In some cases increases in cyclin E2 expression are not associated with proliferation, but instead with other oncogenic attributes such as invasion 100 and drug resistance 101 102 . Cyclin E1 may enhance tumorigenesis through increasing genomic instability, an ability which may be derived from promoting premature replication licensing and endoreplication 96 103 . Specific experiments have not been reported that examine whether genomic instability is also induced through cyclin E2 overexpression. Given that recent evidence seems to identify cyclin E1, but not cyclin E2, as a mediator of endoreplication in hepatocytes 44 , cyclin E1 and E2 may not have equivalent roles in the induction of genomic instability.

Due to the limited availability of antibodies suitable for immunohistochemistry, the expression of cyclin E2 has been examined only through mRNA expression in tumour samples, whereas there is a comprehensive array of literature on the expression of both cyclin E1 protein and mRNA in cancer samples 2 104 . CCNE1 and CCNE2 expression has been compared in breast cancer, where these studies are representative of the majority of studies on the relationship of CCNE1 to breast cancer 104 . Both CCNE1 and CCNE2 are expressed at higher levels in breast cancer, with an association of both genes to increased tumour grade 105 106 , estrogen receptor (ER) negative status 105 106 , progesterone receptor negative status 106 , and proliferative index by Ki67 staining 83 105 . High levels of CCNE1 have a more significant relationship than CCNE2 with each of these parameters. Both CCNE1 and CCNE2 have an inverse linear relationship between expression and metastasis-free survival, which is particularly strong for CCNE2 107 , and high expression of each E-cyclin mRNA also predicts poor overall survival 106 and shorter relapse-free interval 105 . One study also found some correlation between the expression of CCNE1 and CCNE2 83 , although each E-cyclin is independently corrleated to poor overall and metastasis-free survival 106 . CCNE2 is also a component of three prognostic gene expression signatures that predict shorter metastasis-free survival or relapse-free survival of breast cancer patients, whereas CCNE1 does not feature in any of these signatures 108 109 110 .

Differences become apparent between CCNE1 and CCNE2 in their relationship to survival and response to therapy of ER positive and anti-estrogen (tamoxifen) treated patients. High CCNE1 expression predicts shorter metastasis-free survival in both ER negative and ER positive patients, whereas CCNE2 expression is only predictive for the ER positive patient subset 105 106 . By contrast, in primary tumours high levels of CCNE1, but not CCNE2, predict a shorter relapse-free interval of tamoxifen-treated patients 105 . However CCNE2 has been detected at high levels in recurrent disease after tamoxifen treatment 111 , hinting at a functional, if not prognostic, role. In tamoxifen resistant cell lines, CCNE2, but not CCNE1, is induced as part of the agonist response to tamoxifen 112 . Anti-estrogen resistance in MCF-7 breast cancer cells conferred by the TNFα inhibitor, A20, is also associated with increases in cyclin E2 expression 113 . This is consistent with studies in estrogen-responsive breast cancer cell lines where cyclin E2 is a highly estrogen responsive target, whereas cyclin E1 is only marginally increased 75 114 .

CCNE1 is expressed at high levels in multiple tumour types other than breast 2 . CCNE2 shows moderate increases in expression in various malignancies, such as lung, ovarian, nasopharyngeal, colorectal, non small cell lung cancer and leukaemia 83 88 115 116 117 , and these increases in expression are frequently not correlated to CCNE1 expression 83 . In two studies the expression of CCNE2 is lower in ovarian and non small cell lung tumours than matched controls, whereas CCNE1 expression remains unchanged or increases 118 119 . Two further studies indicate that CCNE2 is expressed at significant levels in recurrent disease, including therapy-related myeloid leukemia 120 and recurrent non small cell lung carcinoma 121 . These data reflect the more comprehensive data collected with respect to breast cancer, where CCNE2 expression is frequently upregulated in tumours independently of CCNE1, and CCNE2 is often detected in recurrent disease.

Cyclin E1 and E2 display largely overlapping functions, and high redundancy. Does the presence of two E-cyclins provide a safety net to ensure proliferative potential, or do they have unique functions? The E-cyclin gene pair has been maintained without significant divergence throughout the evolution of higher eukaryotes, implying a selective pressure for the maintenance of each gene. Genome wide studies in yeast have found that apparently redundant paralogs do in fact have distinct and non-overlapping functions, although these only become evident in circumstances of stress or particular stimuli 122 . This is certainly the case for cyclin E1 and E2, where cyclin E1 promotes hepatocyte endoreplication after liver injury, whereas cyclin E2 suppresses this process 44 . Further differences between cyclin E1 and cyclin E2 are possibly found during meiosis and embryogenesis 13 39 . There are tantalising observations on the activity of cyclin E in Drosophila and C. elegans which suggest that there may be additional functions for the E-cyclins in DNA replication of embryonic cells and lineage determination of stem cells. Consequently the differences between cyclin E1 and E2 may be more apparent in non-mitotic cell cycles than in normally proliferating cells, which may explain their apparent redundancy in common laboratory model systems.

Cancer cell cycles represent another form of aberrant cell division, often compared to embryonic cell cycles, and cyclins E1 and E2 promote oncogenic transformation and to promote cancer cell proliferation 13 . Data from cancer studies have already identified that cyclin E1 and E2 are frequently not co-expressed in tumours, and may have distinct associations with recurrent disease. A likely explanation for the discordant expression of cyclin E1 and E2 in cancer is their regulation by distinct subsets of transcription factors and miRNAs. The expression of cyclin E2, independently of cyclin E1, can be induced via cyclin D1, Chd8 and CDP/Cux, all of which are upregulated in cancers 47 123 , and cyclin E2 is also independently suppressed by the tumour suppressor p53 89 . The co-regulation of cyclin E1 and cyclin E2 with distinct gene sets could explain the different relationship of each gene to disease. For example, cyclin E2, which is a target of cyclin D1 and Chd8 in estrogen-responsive cells 75 , has been associated with poor outcome only in ER-positive breast cancers 106 , which resembles the association of cyclin D1 overexpression to ER-positive but not ER-negative breast cancers 124 . The functional outcome of overexpression of cyclin E1 compared to cyclin E2 is not known. However, given the potential differences in function in endoreplication and other processes, cyclin E1 and cyclin E2 may have distinct attributes that contribute to cancer progression.

The cyclin and Cdk proteins of the cell cycle have considerable redundancy and compensation between members of each family 125 , yet careful study reveals unique functions of many of these proteins. For example, cyclin D2 knockin cannot completely substitute for cyclin D1 in mouse development 126 . Recent data on the function and regulation of cyclin E1 and cyclin E2 demonstrates that they are also not redundant homologs (Table 1). The identification of cyclin E1 as promoting, and cyclin E2 as repressing, hepatocyte endoreplication deserves further investigation to unravel the mechanistic difference between the two proteins. Cyclin E1 and E2 should also be investigated as distinct entities in cancer studies, especially in the context of examining relationships with other markers of tumorigenesis such as cyclin D1 and p53. There will be difficulties in ongoing studies on cyclin E1 and cyclin E2 as they do have considerable functional redundancy in their interactions with Cdk2 and Cdk inhibitor proteins, as well as having the potential to regulate one another. The identification of further differences between the E-cyclins is likely to require an appropriate molecular environment that highlights their differences, such as during endoreplication or the proliferation of cancer cells.

CCNE1: Cyclin E1 mRNA; CCNE2: Cyclin E2 mRNA; Cdk: cyclin dependent kinase; CLS: centrosome localisation sequence; ER: estrogen receptor; miRNA: microRNA; Pre-RC: pre replication complex; TSC: trophoblast giant cell.

The authors declare that they have no competing interests.

CC and EM drafted the manuscript. Both authors read and approved the final manuscript.