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Z.E.WINTERS
University Division of Surgery, University of Bristol, Bristol Royal Infirmary, Malborough Street, Bristol BS2 8HW, UK.
King James IV lecture delivered at BASO, Glasgow, 27 November 2001
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Keywords: G2 checkpoint; cyclin B/Cdc2; p53
J.R.Coll.Surg.Edinb., August 2002, 591- 598
DNA damage activates checkpoint pathways to produce a G1 or G2 cell cycle arrest and DNA repair. G2 checkpoint integrity prevents inappropriate mitosis of unrepaired DNA. Cell cycle progression is determined by cyclin-dependent kinase (CDK) enzymes in association with specific cyclin proteins, with Cdc2/cyclin B regulating mitosis. The tumour suppressor p53 re-enforces G2 arrest through the CDK inhibitor, p21.WAF1/CIPI Functional regulation of G2 checkpoint proteins occurs through levels of protein expression, phosphorylation and subcellular localisation
Eukaryotic cells regulate growth through complex signalling pathways that act to maintain the co-ordinated and integrated sequence of DNA replication (DNA synthesis, S-phase), that precedes mitosis in the cell cycle.1 Cyclin-dependent kinase enzymes (CDKs) determine cell cycle progression, such that their activation depends on an association with a phase-specific cyclin protein. (Figure 1) DNA damage activation of ‘checkpoints’ ensures genomic integrity through inhibition of CDKs to effect a cell cycle arrest and repair prior to replication (G1 checkpoint), or mitosis (G2 checkpoint), with apoptosis constituting an alternative pathway of eliminating DNA damaged cells.2-4
Loss of checkpoint function is a hallmark of many human cancers where there is replication and segregation of damaged DNA. The p53 tumour suppressor protein, which is frequently mutated in human cancers, has an established role in the G1 checkpoint, with accumulating evidence to support its significance in sustaining a damage-induced G2 arrest.5-7 Entry into mitosis requires the association of the G2 cyclin; cyclin B1 with Cdc2, the prototypical CDK, to activate Cdc2 protein kinase. Genotoxic stresses stabilise p53 protein allowing it to function as a nuclear transcription factor that induces expression of several target genes including p21,WAF1/CIPI Bax and Mdm2.8,9 (Figure 2) p21WAF1/CIPI functions as a universal cyclin-dependent kinase inhibitory (CDKI) protein, with an affinity for G1 and G2 cyclin-CDK complexes, underscoring a key mechanism for p53-mediated G1 and G2 cell cycle arrest, in which p21WAF1/CIPI may assemble and target the cyclin-CDK complex to the nucleus prior to inhibition.7,10 The p21WAF1/CIPI-dependent G1 inhibition of CDK complexes functions through activating the retinoblastoma protein (pRb) to repress the E2F-dependent transcription of S-phase genes and produce a G1/S arrest.11 Similarly, pRb has been shown to function in concert with p21WAF1/CIPI to reinforce the G2 checkpoint and ensure a blockade in inappropriate DNA replication.12,13 Mdm2 functions downstream of p53 to regulate its activity in an autoregulatory feedback loop.14 p53-induced Mdm2 inhibits p53 transcription and degrades p53 through a ubiquitin-dependent proteolysis which requires the nuclear export of both p53 and Mdm2.15,16 A nuclear protein ARF (Alternative Reading Frame) is one of two alternatively spliced transcripts; the other being the p16 G1 CDKI protein encoded by the INK4A-ARF locus.17 Human p14ARF functions upstream of p53 in the same tumour suppressor pathway, to stabilise active p53 in the nucleus, secondary to ARF binding Mdm2 to inhibit the latter’s activity and nucleocytoplasmic shuttling.16, 18 ARF over-expression may in part re-enforce p53-induced G1 and G2 arrest. (Figure 3) Other G2 checkpoint regulatory pathways inhibiting cyclin B1-Cdc2 activity involve regulatory phosphorylation, subcellular localisation, and expression of cyclin B1 and Cdc2 proteins. Nucleocytoplasmic shuttling of cell cycle regulatory proteins enables ‘cross-talk’ between nuclear and cytoplasmic compartments. Specific amino-acid sequence motifs determine the nuclear localisation signal (NLS), with a leucine-rich nuclear export signal (NES) governing interaction with the CRM1 nuclear export receptor.19,20
Mitosis and cellular proliferation depend on activation of Cdc2.21 This is associated with increased levels of cyclin B and Cdc2, with enhanced cyclin binding, the removal of inhibitory phosporylation of Cdc2 by Cdc25 phosphatase, and the nuclear localisation of the cyclin B/Cdc2 complex. p53 dampens cyclin B/Cdc2 kinase activity by influencing these fundamental pathways of G2 checkpoint control.6,7,22 Activated p53 downregulates cyclin B and Cdc2 expression through transcriptional repression, in a pathway dependent on p21WAF1/CIPI and pRb.13,23,24 An association of p21WAF1/CIPI with cyclin B/Cdc2 inhibits kinase activity to prevent phosphorylation and consequent inhibition of pRb. Active hypophosphorylated pRb binds E2F to restrict E2F-mediated transcription of cyclin B and Cdc2. Abnormalities in this pathway, may partly explain the relationship between increased cyclin B/Cdc2 levels and tumour invasion and aggressiveness.25-27
Figure 1: DNA damage activation of cell cycle checkpoints: Cell cycle progression is regulated by phase-specific cyclin-dependent kinase (CDK) enzymes whose activity depends on an association with a specific cyclin protein. Cyclin B/Cdc2 kinase activity drives the G2/M phase transition. G1/S phase arrest depends on the retinoblastoma protein (pRb) binding the E2F transcription factor. p16 is a G1 cyclin-dependent kinase inhibitor
Figure 2: p53 re-enforces G1 and G2 cell cycle arrest after DNA damage through the cyclin-dependent kinase inhibitor p21WAF1/CIPI Mdm2 and Bax are other p53 transcriptional targets, with Mdm2 regulating p53 levels and Bax mediating apoptosis
Figure 3: DNA damage activation of G2 checkpoint regulators. p53 participates in an auto-regulatory feedback loop with Mdm2 and p14ARF pRb and p21WAF1/CIPI regulate cyclin B-Cdc2 kinsase activity to re-enforce G2/M cell cycle arrest
The cyclin B/Cdc2 complex undergoes nucleocytoplasmic shuttling as a means of co-ordinating important nuclear and cytoplasmic functions (Figure 4). The subcellular localisation of cyclin B/Cdc2 and its regulatory molecules may constitute an important aspect of regulation governing mitotic entry and influencing DNA damage-induced checkpoint control. Hence, cyclin B is cytoplasmic during S and G2 phases, with a nuclear translocation required to initiate mitosis.28 Cytoplasmic cyclin B may result from an anchoring effect conferred by a cytoplasmic retention signal (CRS) and/or a rapid nuclear export by a CRS behaving as a nuclear export signal (NES) to facilitate a cyclin B interaction with CRM1.29,30 By contrast, cyclin B/Cdc2 localises to the nucleus through mechanisms involving the import receptor; importin ß and/or phosphorylation of the CRS/NES to interfere with CRM1 binding and nuclear export.31 G2 checkpoint pathways have the potential to inactivate cyclin B/Cdc2 kinase regardless of its nuclear or cytoplasmic localisation, by mechanisms depending on cell type and functional p53. Evidence strongly suggests that active p53 prolongs G2 arrest through the nuclear accumulation of p21WAF1/CIP1 in association with cyclin B/Cdc2.7,10 Further complexity is suggested by an alternative p53 pathway transcriptionally activating the 14-3-3s protein which delays mitosis after DNA damage by anchoring cyclin B/Cdc2 in the cytoplasm in a pathway that may complement p21WAF1/CIPI mediated nuclear inhibition of Cdc2 kinase.32,33 Separate pathways of Cdc2 inhibition include inhibitory phosphorylation by nuclear Wee1 kinase and cytoplasmic Myt1 kinase.34 14-3-3s binds Wee1, with the potential to facilitate a Cdc2-Wee1 interaction. Other 14-3-3 proteins (excluding 14-3-3s) independent of p53, maintain Cdc2 kinase inhibitory phosphorylation by inactivating nuclear Cdc25 phosphatase, which they sequester in the cytoplasm. p53 contributes to a sustained G2 arrest through cyclin B/Cdc2 inhibition by co-localisation of the complex with either nuclear p21WAF1/CIPI or cytoplasmic 14-3-3s. Conversely, p53-independent G2 arrest is transient, with a preferential cytoplasmic localisation of cyclin B/Cdc2, and Cdc2 kinase inhibition by inhibitory phosphorylation.7 Accordingly, predominant cytoplasmic cyclin B/Cdc2 may characterise increased tumour recurrence following radiotherapy.35
p53-MDM2-ARF
As a tumour-suppressor, p53 requires the nuclear localisation and transcriptional activation of target genes to produce cell cycle arrest and apoptosis in a process eliminating aberrant cells. Potential dysregulation in any of these steps may produce carcinogenesis. Elements regulating p53 function may include factors influencing its subcellular localisation. Cytoplasmic p53 found in some human tumours may reflect diminished efficiency of nuclear accumulation of p53 and provide a mechanism for carcinogenesis.36-38 p53 is actively transported between the cytoplasm and nucleus.39 Nuclear import is determined by interaction with the cytoplasmic microtubule network and facilitated through the carboxyterminal nuclear localisation signals (NLS).40,41 A nuclear export signal in the tetramerization domain of p53, potentially influences p53 function by either facilitating nuclear export to dampen p53 activity, or blocks nuclear export to enhance p53 effect.42,43 Critical to the regulation of p53 is its interaction with Mdm2.44,45 As a negative p53 regulator, Mdm2 contributes to the short half-life and low levels of p53 in normal cells, with a role as an oncoprotein in certain tumours.46 Mdm2 degrades p53 in its role as a ubiquitin protein ligase with evidence to suggest that polyubiquitinated p53 undergoes nuclear export and Mdm2-targeted degradation in the cytoplasmic proteosome.47 Nuclear export of Mdm2 is influenced by its own ubiquitination and nuclear export signal, in association with a constant shuttling via its nuclear import signal.42 Evidence supports a number of models influencing p53 nuclear export and inactivation including; the necessary transport (both nuclear import and export) of Mdm2 throughout the cell, the nuclear ubiquitination of p53 prior to its independent nuclear export,48 or the need for the simultaneous nuclear export of Mdm2 together with p53.16 Cellular insults such as DNA damage, hypoxia and oncogene stimulation elevate nuclear p53 through processes that prevent p53 and Mdm2 interaction, and through p14ARF that inhibits the ubiquitin-ligase activity of Mdm2. Mutations of the INK4A-ARF and p53 loci occur with a similar frequency in cancer cells. AFR localises predominantly to the nucleolus through its carboxy-terminal nucleolar localisation signal but is also found within nucleoplasm.49-51 As a tumour suppressor, ARF stabilises p53 in the nucleus through a number of possible-mechanisms. ARF could sequestrate Mdm2 away from p53, binding Mdm2 in the nucleolus and in the nucleoplasm to inhibit the nuclear export of Mdm2, with a resultant decrease in p53 nuclear export and cytoplasmic degradation. Nucleolar or nucleoplasmic ARF-Mdm2 may be important in another mechanism of Mdm2 inhibition, namely ARF inhibition of Mdm2 ubiquitin-ligase activity. It is unlikely that an exclusive nucleolar ARF-Mdm2 is required to stabilise p53. The influence of ARF and Mdm2 on the subcellular localisation of p53 and its function underlines the importance of evaluating these components in the p53 pathway.
Figure 4: Cyclin B/Cdc2 kinase activity is regulated through nucleocytoplasmic shuttling, cyclin B/Cdc2 undergoes nuclear import prior to mitosis via importinß and is rapidly exported into the cytoplasm through the CRM1 nuclear export receptor. Leptomycin B (LMB) identifies CRM1 specificity. Cdc2 kinase activity is dampened by Myt1 and Wee1 inhibitory kinases in the cytoplasm and nucleus, respectively. Stimulatory phosphatases include cytoplasmic Cdc25B, and nuclear Cdc25C
p21WAF1/CIPI
p21WAF1/CIPI has a role in cell cycle arrest, to effect cellular differentiation and senescence following growth factor stimulation and DNA damage. A tumour suppressor function can be inferred by its ability to inhibit oncogenic transformation. As a cell cycle regulator, p21WAF1/CIPI shows a bimodal periodicity-with peaks in G1 and G2/M.52 DNA damage activation of p53 increases p21WAF1/CIPI to sustain a G1/S and G2/M arrest, compared to the transient cell cycle arrest in cells lacking p21.WAF1/CIPI 6,7,53 CDK inhibition by p21WAF1/CIPI is correlated with its nuclear localisation, which is determined by a carboxy-terminal NLS.54 Nuclear accumulation of p21WAF1/CIPI appears integral to its checkpoint control by its ability to confer a nuclear localisation on cyclin B/Cdc2 to decrease Cdc2 kinase activity and sustain G2 arrest. 7,10 Increased p21WAF1/CIPI expression in cancers may be associated with increased proliferation to suggest an inability of p21WAF1/CIPI to affect cell growth.55-57 Inactivation of p21WAF1/CIPI rarely includes mutationsbut could relate to changes in subcellular localisation. Recent evidence has demonstrated the cytoplasmic localisation of p21WAF1/CIPI in cancer tissues and cell lines.55 Potential abnormalities in p21WAF1/CIPI nuclear localisation may occur through pathways either truncating the carboxy-terminus to eliminate the NLS, or through phosphorylation of the NLS to produce a cytoplasmic distribution.58,59
C-erbB2 overexpression activates the phosphatidylinositol-3 kinase (PI-3K) pathway to phosphorylate the p21WAF1/CIPI NLS and induce cytoplasmic p21WAF1/CIPI.59 (Figure 5) A novel form of p21WAF1/CIPI through loss of carboxy-terminal amino acids is cytoplasmic and characterises a tumourigenic cell line.58 Cytoplasmic p21WAF1/CIPI may inhibit apoptotic death by bindingand inhibiting the apoptosis signal-regulating kinase 1 (ASK1) and the subcellular localisation of p21WAF1/CIPI would be important in this function.56
Figure 5: HER2/neu blocks apoptosis in breast cancer by localising p21WAF1/CIPI in the cytoplasm, PI-3 --phosphatidylinositol-3 kinase; Akt - kinase downstream of PI3; ASK1- Apoptosis Signal Regulating Kinase 1
Dysregulated levels of protein expression and subcellular localisation may influence cell cycle progression resulting in autonomous cellular proliferation and neoplastic transformation. Alterations in cyclin B and Cdc2 are a widespread feature of tumourigenesis. Proportionately increased expression of cyclin b and Cdc2 in breast cancer correlates with mitotic and/or ana-telophase index, and with an increased incidence of lymph node metastasis and high grade tumours.60 Immunohistochemical documentation of cytoplasmic or nuclear cyclin B independently predict a poor prognosis.57
Impaired p53 function through mutations in the gene can occur in 42% of mammary carcinomas.61 This variably correlates-with immunohistochemical detection of p53, its ability to transactivate target genes and to localise in the nucleus. Stabilisation of p53 due to failed transactivation of Mdm2 may produce the p53 overexpression detected immunohistochemically which was found to be an independent prognostic marker in 32% of studies.62 This is, however, not consistent with those mutations that hasten p53 degradation, or in conditions of homozygous deletion where there is no p53 production. The relevance of cytoplasmic p53 as a prognostic marker remains to be clarified since a recent p53 mutant with defective nuclear localisation transactivates p53 responsive genes and induces apoptosis.63
Neoplasia is associated with complex pathways between p53 and its downstream transcripts; p21WAF1/CIPI and Mdm2, with both wild-type and mutant p53 being associated with variable expression of p21WAF1/CIP and Mdm2. This may relate to p53-independent pathways of induction, and the potential for Mdm2 gene amplification found in 5% of breast cancers.64,65
Subcellular mislocalisation of p21WAF1/CIPI may reflect abnormal p53 and predict prognosis in breast cancer since cytoplasmic p21WAF1/CIPI occurs as a consequence of c-erbB2 overexpression.57,59 A failure of Mdm2 nuclear localisation may occur following ARF inactivation and decreased p14ARF expression.66 ARF loss of heterozygosity or methylation correlates with several poor prognostic parameters in breast cancer, and frequently co-exists with p53 mutations.67 Any adverse prognostic influence of cytoplasmic Mdm2 requires evaluation. A recently identified cytoplasmic protein Pex19p, that binds ARF, has been shown to inactivate p53 by excluding p14ARF from the nucleus.68
The tumour suppressor role of p53 requires its nuclear activation and retention. Nongenotoxic strategies directed to this end may include; disruption of p53-Mdm2 complexes by antibodies, artificial constructs, or antisense inhibition of Mdm2 expression.20,54 Potential molecular pathways blocking the Mdm2 inhibitory effect on p53, involve the c-Abl nonreceptor tyrosine kinase, and the MdmX protein, which unlike Mdm2 stabilises and transactivates nuclear p53.69,70 A recently identified synthetic p14ARF amino-terminal peptide binds Mdm2 to inhibit its ubiquitinating ability, so activating p53 in vivo.71 Other mechanistic approaches attempt to block p53 nuclear export, through the proteosome inhibitor; lactacystin, and through Leptomycin B which inhibits the Mdm2 NES to influence p53/Mdm2 nuclear export.72, 73
The p53-mediated effects of apoptosis, DNA repair and checkpoint control at G1, S phase and G2 are potentially important determinants of tumour sensitivity to DNA damage. A number of studies have demonstrated the association of p53 inactivation with sensitisation to DNA damage, through relaxation of G2 checkpoint control, which correlated with enhanced cytotoxicity when cells were exposed to combinations of DNA damaging agents and checkpoint-inhibitory drugs. This suggests the exciting possibility that such combinations could be used therapeutically to target cancer cells specifically, since p53 function is compromised or absent in the majority of human tumours.
p21WAF1/CIPI as a principal mediator of p53 function and a determinant of G2 checkpoint integrity may attenuate druginduced apoptosis by inducing cell cycle arrest and DNA repair. Pathways upregulating p21WAF1/CIPI increase resistance to Paclitaxel (Taxol)-induced apoptosis, through c-erbB2 overexpression.74 Taxol activation of Cdc2 kinase leading to apoptosis is blocked by p21WAF1/CIPI inhibition of cyclin B/Cdc2 kinase. p21WAF1/CIPI may confer protection against the cytotoxicity of radiation and doxorubicin.75,76
Defining checkpoint pathways and their regulation is fundamental to our understanding of how cell stresses may initiate carcinogenesis. Principal components are regulated through levels of protein expression, their degradation and subcellular localisation, which are likely to be interrelated. Translating these in vitro findings to clinical studies provides the basis for promising therapies of the future.
The author is grateful to the late John Farndon, Chris Norbury and Jeff Holly for their advice and comments on the manuscript and to Ms Liz Humphries for typing the manuscript.
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Copyright 17 April 2002
Correspondence: Z.E. Winters, University Division of Surgery,University of Bristol, Bristol Royal Infirmary, Malborough Street, Bristol BS2 8HW, UK