Modulazione farmacologica del ciclo cellulare

Roles of cyclin-dependent kinases Roles of cyclin-dependent kinases. (a) Cyclin-dependent kinases (CDKs) catalytic subunits and cyclins responsible for progression through the indicated cell-cycle phase. Arrowheads indicate activation, and flat bars indicate inhibition by the specific endogenous negative regulator. (b) Transcription-related CDKs promote elongation of nascent transcripts by phosphorylating the carboxy-terminal domain (CTD) of RNA polymerase II. (c) CDK5/p25 facilitates secretion of insulin by phosphorylating an as-yet incompletely defined set of substrates, possibly including munc 18, synapsin-1, and syntaxin, all of which are responsible for secretory vesicle alignment with, for example, the cytoskeleton and membrane (described in detail in [17]).

Interphase cyclin-dependent kinases(CDKs) drive the cell out of quiescence (G0) and beyond the restriction point, resulting in the cell being irreversibly committed to the DNA synthesis (S) phase transition. The DNA-damage checkpoint kinases (CHKs) act as sensor proteins that can induce cell cycle arrest in the first gap (G1) and G1–S phases in response to DNA lesions. CDK1, CDK2, polo-like kinase 1 (PLK1) and aurora A are involved in the regulation of the centrosome cycle, whereas kinases that are involved in the spindle assembly checkpoint pathway ensure proper DNA segregation during mitosis (M) phase. Different kinases may act at several stages of the cell cycle and modulate the activities of other cell-cycle-related kinases. Cancers associated with genetic alteration of specific kinases are indicated in blue boxes. ATM, ataxia telangiectasia mutated (also known as serine protein kinase ATM); ATR, ataxia telangiectasia and RAD3-related protein (also known as serine–threonine protein kinase ATR); BUB1, budding uninhibited by benzimidazoles 1; BUB1B, BUB1 homologue beta (also known as BUBR1); MPS1, monopolar spindle 1; MVA, mosaic variegated aneuploidy; PCSS, premature chromatid separation syndrome; RB, retinoblastoma protein family members.

Regulation of G1 and the G1/S transition Regulation of G1 and the G1/S transition.   In quiescent, G0 cells, E2F–DP transcription factors are bound to p130, the principal pocket protein in these cells, which keeps them inactive. In G1, however, RB–E2F–DP complexes predominate. Mitogenic signalling results in cyclin D (Cyc) synthesis, formation of active CDK4/6–cyclin-D complexes and initial phosphorylation of RB. Partially phosphorylated RB still binds to E2F–DP, but the transcription factor is still able to transcribe some genes, such as cyclin E, presumably due to impaired repression. Cyclin E binds to and activates CDK2. It is generally accepted that CDK2-dependent phosphorylation of RB results in its complete inactivation, which allows induction of the E2F-responsive genes that are needed to drive cells through the G1/S transition and to initiate DNA replication. INK4 and WAF1/KIP proteins can inhibit CDK4/6 or CDK2 kinases, respectively, following specific antimitogenic signals. The CDK4/6 complexes can also bind WAF1/KIP inhibitors, while remaining active. This sequesters them from CDK2, which facilitates its full activation. R represents the restriction point that separates the mitogen-dependent early G1 phase from the mitogen-independent late G1 phase.

Members of the retinoblastoma (RB) protein family, comprising RB1 (also known as p105-RB), retinoblastoma-like protein 1 (RBL1; also known as p107) and RBL2 (also known as p130), share sequence homology in a bipartite domain known as the pocket domain, which folds into a globular pocket-like structure owing to the presence of a flexible 'spacer' region. The pocket domain mediates interactions with members of the E2F family of transcription factors and with proteins containing an LXCXE motif, such as D-type cyclins (CYCDs), histone deacetylases and viral oncoproteins. RB family members, or 'pocket proteins', play key parts in the control of cell proliferation. They negatively modulate the transition from the first gap (G1) phase to the DNA synthesis (S) phase (see figure), are growth-suppressive in a cell type-dependent manner, are implicated in various forms of differentiation and are crucial targets for inactivation by transforming oncoproteins of DNA tumour viruses3, 111. The G1 to S phase transition is a complex process, involving the concerted actions of various cyclins and cyclin-dependent kinases (CDKs) in conjunction with the RB proteins (see figure). Mitogenic stimuli induce the release of the cyclin D-associated kinases, CDK4 and CDK6, from the inhibitory INK4 proteins (p16INK4A, p15INK4B, p18INK4C and p19INK4D) and initiate phosphorylation of RB1, RBL1 and RBL2. Cyclin D–CDK4 and cyclin D–CDK6 complexes also bind stoichiometrically to the potent CDK2 inhibitors of the Cip–Kip family (p27 (also known as CDKN1B and KIP1), p21 (also known as CDKN1A and CIP1) and p57 (also known as CDKN1C and KIP2)), sequestering them away from CDK2. Partially phosphorylated RB proteins release E2F transcription factors, enabling the expression of genes required for G1 to S phase transition and DNA synthesis. This includes the cyclin E gene, the protein product of which binds and allosterically regulates CDK2 activity in late G1, creating a positive feedback loop that antagonizes Cip–Kip inhibitors by signalling for their proteolysis, and reinforces RB inactivation, leading to an irreversible switch to S phase. Cyclin A- and cyclin B-dependent CDKs are activated at later phases of the cell cycle to maintain RB in a hyperphosphorylated form until the cell exits mitosis. RB family members112 and Cip–Kip proteins113 may also be involved in maintaining cells in a quiescent state (G0). The functional overlap between the RB proteins does not seem to extend to complete redundancy. First, their expression levels differ in the various phases of the cell cycle: RBL2 expression is higher in the G0 phase, RBL1 expression peaks during the S phase, and RB1 expression is uniform throughout the cell cycle. Second, their expression is cell type-specific: RBL2 predominates in neurons and skeletal muscle, RBL1 expression is particularly high in breast and prostate epithelial cells, whereas RB1 is ubiquitously expressed in normal tissues112. Third, each of the pocket proteins interacts with specific subsets of E2F transcription factors, although overexpression of any RB family member causes cell cycle arrest in G1 in most cell types. Fourth, RB1, but not RBL1 or RBL2, can bind to the anaphase-promoting complex or cyclosome and stabilize p27, promoting cell cycle exit114. Finally, RBL1 and RBL2, but not RB1, associate stoichiometrically with either cyclin E–CDK2 or cyclin A–CDK2 complexes, thereby inhibiting the complexes115, 116. RBL1 is thought to act as an E2F competitor by binding to CDK2–cyclin complexes through its amino-terminal region. By contrast, RBL2 could act as a direct CDK2 inhibitor117, 118. Such activity was found to be mediated by the spacer region of RBL2, which has an amino-acid sequence that is unique among the other members of the RB family119. Retinoblastoma, a relatively rare cancer, has dramatically changed the way cancer is studied and understood, through important scientific advances such as the identification of RB1 as the first tumour suppressor gene. Currently, loss of RB1 is considered either a causal or an accelerating event in many cancer types120, 121. Depending on the tumour type, loss of RB1 function is associated with different responses to various therapeutic agents122. By contrast, much less is known regarding the tumour-suppressive functions of RBL2 and RBL1, which are less frequently inactivated in human tumours compared with RB1. Several lines of evidence show that functional inactivation of RBL2 or RBL1 can provide a growth advantage during later stages of cancer123. A thorough grasp of the role of the RB gene family in cancer remains a challenge. Further studies to elucidate the relationships between the status of all members of the RB family of proteins and the response to different treatments in a cancer type-specific context will be crucial for accurate prognosis and optimal treatment. M, mitosis phase.

REGOLAZIONE DELL’ATTIVITÀ DELLE CHINASI CICLINA-DIPENDENTI sintesi/degradazione delle relative cicline formazione/dissociazione di complessi con inibitori (p21waf1/cip1; p27kip1; p16ink4A) fosforilazione/defosforilazione di Tyr15/Thr14 fosforilazione/defosforilazione T-loop (Thr160/161)

a | Cell division cycle 25 (CDC25) phosphatases dephosphorylate and activate cyclin-dependent kinase (CDK)–cyclin complexes, thus allowing catalysis and substrate phosphorylation. WEE1 and MYT1 kinases phosphorylate CDK on tyrosine 15 and threonine 14 of CDK1. Phosphorylation by the CDK-activating kinase (CAK) is required for further activation of CDK–cyclin complexes. For simplicity, an orange P represents T14 and Y15 phosphorylation by WEE1 and MYT1, a blue P represents T161 phosphorylation by CAK.

Mutation of G1/S regulators in human cancer Mutation of G1/S regulators in human cancer.   Only alterations that occur in more than 10% of primary tumours have been considered. Numbers represent the percentage of tumours with alterations in any of the listed cell-cycle regulators. The loci in which specific genetic or epigenetic alteration have been defined are in pink. The alterations for which no mechanistic explanation has been provided are in yellow. Alterations relevant for tumour prognosis are indicated by asterisks.

Generations of cyclin-dependent kinase inhibitor compounds Generations of cyclin-dependent kinase inhibitor compounds. The `pan' cyclin-dependent kinase (CDK) inhibitor flavopiridol potently inhibits all known CDKs to an approximately equal degree. It possibly has a greater potency for CDK9 owing to the capacity to form an ultra-tight binding complex. This has inspired design efforts to derive a second generation of agents with a spectrum of selectivity for the indicated kinase.

(ALVOCIDIB)

MECCANISMI D’AZIONE DEL FLAVOPIRIDOLO Inibizione EGFr (IC50 = 21 M) Inibizione PKA (IC50 = 122 M) Inibizione proliferazione cellulare (IC50 = 66 nM) Inibizione CDKs cdk1, cdk2, cdk4, cdk6 (IC50 = ~ 41 nM)

Transformed cells are sensitized to a cdk2 inhibitor during S phase Transformed cells are sensitized to a cdk2 inhibitor during S phase. During normal cell cycle progression (top), cyclin A–cdk2 phosphorylates E2F-bound DP-1 in order to downregulate E2F at the appropriate time and allow orderly S phase progression. Following recruitment to S phase by chemotherapy agents (bottom), inhibition of cyclin A–cdk2 prevents E2F-1/DP-1 phosphorylation, resulting in inappropriately persistent E2F-1 activity and eventual apoptosis.

MECCANISMI D’AZIONE DEL FLAVOPIRIDOLO Inibizione EGFr (IC50 = 21 M) Inibizione PKA (IC50 = 122 M) Inibizione proliferazione cellulare (IC50 = 66 nM) Inibizione CDKs cdk1, cdk2, cdk4, cdk6 (IC50 = ~ 41 nM) cdk7 (IC50 = 300 nM)

MECCANISMI D’AZIONE DEL FLAVOPIRIDOLO Inibizione della trascrizione (IC50 > 100 nM)

Ciclina D1 XIAP Bcl2 Mcl1 etc. Flavopiridol Roles of cyclin-dependent kinases. (a) Cyclin-dependent kinases (CDKs) catalytic subunits and cyclins responsible for progression through the indicated cell-cycle phase. Arrowheads indicate activation, and flat bars indicate inhibition by the specific endogenous negative regulator. (b) Transcription-related CDKs promote elongation of nascent transcripts by phosphorylating the carboxy-terminal domain (CTD) of RNA polymerase II. (c) CDK5/p25 facilitates secretion of insulin by phosphorylating an as-yet incompletely defined set of substrates, possibly including munc 18, synapsin-1, and syntaxin, all of which are responsible for secretory vesicle alignment with, for example, the cytoskeleton and membrane (described in detail in [17]). Ciclina D1 XIAP Bcl2 Mcl1 etc.

MECCANISMI D’AZIONE DEL FLAVOPIRIDOLO Inibizione della trascrizione (IC50 > 100 nM) Arresto in G1 Possibile effetto anti-HIV? Apoptosi

MECCANISMI D’AZIONE DEL FLAVOPIRIDOLO Inibizione dell’angiogenesi (IC50 = 50-100 nM) Destabilizzazione del mRNA per VEGF

EFFETTI TOSSICI DEL FLAVOPIRIDOLO Diarrea secretoria Ipotensione Sindrome proinfiammatoria reversibili

Induced or spontaneous DNA lesions are common events in the life of the cell. The ability of the cell to maintain homeostasis and protect itself from neoplastic transformation depends upon complex surveillance mechanisms and activation of repair pathways to preserve chromosomal integrity. The DNA damage checkpoint is a cardinal process. Genetic defects that perturb DNA repair mechanisms almost always cause severe diseases, including ataxia-telangiectasia and related syndromes, characterized by degeneration of the nervous and immune systems, sensitivity to ionizing radiation and DNA-damaging agents, and predisposition to cancer. The serine–threonine protein kinases ataxia telangiectasia mutated (ATM; also known as serine protein kinase ATM) and ataxia telangiectasia and RAD3-related protein (ATR; also known as serine-threonine protein kinase ATR) are DNA damage sensor proteins that can induce cell cycle arrest, DNA damage repair or apoptosis, depending on the extent of the DNA lesions (see figure). Whereas ATM responds primarily to DNA double-strand breaks, which are generally caused by ionizing radiation and radiomimetic drugs, ATR also responds to damage caused by ultraviolet light and stalled replication forks55. The ATM–ATR cascade is activated within minutes of a DNA damage alarm. Both ATM and ATR can phosphorylate and activate the transcription factor p53, either directly or by means of prior activation of checkpoint kinase 2 (CHK2). Among the genes induced by p53 is the cyclin-dependent kinase 2 (CDK2) inhibitor p21 (also known as CDKN1A and CIP1), the activity of which prevents damaged cells from entering the DNA synthesis (S) phase. Also, damaged cells that have already passed the transition from the first gap (G1) phase to S phase can be halted through the activation of another ATM–ATR effector, CHK1, which phosphorylates the dual-specificity phosphatase CDC25C, providing a signal that induces its sequestration in the cytoplasm. Because CDC25C is responsible for removing two inhibitory phosphates from CDK1, its inactivation prevents the cell from entry into the mitosis (M) phase. Cell cycle arrest in G1, S or G2 phase is maintained until DNA integrity is restored. If lesions are irreparable, programmed cell death is induced by the ATM–ATR signalling pathway The ATM–CHK2 pathway predominantly regulates the G1 checkpoint, whereas the ATR–CHK1 pathway predominantly regulates the S and G2 checkpoints, although there is crosstalk between these pathways. In most human cancers, however, the function of the DNA damage checkpoint in G1 is impaired owing to mutations in p53 or the gene encoding the retinoblastoma protein (RB1). Treatment of these tumour cells with DNA-damaging agents, such as ionizing radiation and DNA-targeting drugs, results in S or G2 checkpoint-mediated arrest. Nonetheless, some of these cells might use this remaining checkpoint to protect themselves from radiation or cytotoxic agents. These cancer-favouring circumstances may be tackled by the combination of DNA-damaging drugs or ionizing radiation with inhibitors of the S or G2 checkpoints, or 'S or G2 checkpoint abrogators'. Such a combination should force cancer cells carrying DNA lesions into mitosis, a condition which prompts mitotic catastrophe and associated cell death. Abrogation of the DNA damage checkpoint in S or G2 is an attractive strategy for selectively targeting G1 checkpoint-defective cancer cells and is currently being explored in clinical trials.

Functions and regulations of CHK2 in the mammalian DNA-damage-response network.   CHK2 is mainly activated by DNA-strand-breaking agents such as ionizing radiation and topoisomerase inhibitors through the ATM-dependent pathway. Other checkpoint proteins, such as 53BP1, MDC1 and the MRE11–RAD50–NBS1 complex, might modulate CHK2 activation (not shown). The role of CHK2 in checkpoints is not totally clear (see main text for details), although it has been shown to phosphorylate CDC25A in vitro, which inhibits its activity. The role of CHK2 in DNA-damage-induced apoptosis is better established. It operates through both p53-dependent and p53-independent — through PML and E2F — pathways. Pifithrin- can block p53-dependent transcription and apoptosis, and has been used as a tool to validate the p53-dependent pathway as a radio/chemosensitization target. CHK2 has also been shown to phosphorylate the BRCA1 protein and might modulate the role of BRCA1 in DNA repair. Red lines represent interactions that are supported by only limited data

Functions and regulations of CHK1 in the mammalian DNA-damage-response network.   CHK1 is activated by a broad spectrum of DNA-damaging agents, including DNA-strand-breaking agents such as ionizing radiation and topoisomerase inhibitors, and those agents that cause replication stress such as ultraviolet light, hydroxyurea and 5-fluorouracil. CHK1 activation that is induced by replication stress is ATR dependent, and its activation by strand-breaking agents is believed to be mainly ATM dependent. Optimal activation of CHK1 also requires other checkpoint proteins such as BRCA1, claspin, the RAD9–RAD1–HUS1 complex and RAD17 (not shown). The role of CHK1 in the S-phase and G2–M checkpoint is best understood and is mediated by phosphorylation of CDC25A and CDC25C, respectively. For simplicity, only one phosphorylation site is indicated for each substrate of CHK1 (though often more than one site is targeted). Other potential CHK1 functions include chromatin remodelling — through the tousled-like kinases (TLK1/2) — replication and DNA repair. UCN-01 is a potent CHK1 inhibitor and has been used as a tool compound to study CHK1-dependent pathways. Dashed lines represent less strong interactions. Red lines represent interactions that are supported by only limited data.

Cells are constantly subjected to DNA damage and DNA breaks that arise either endogenously during normal cellular processes such as genome replication or exogenously by exposure to genotoxic agents, such as UV radiation, radiotherapeutics and chemotherapeutics. Damage to the DNA triggers the recruitment of specific damage sensor protein complexes. On the one hand, the MRN (MRE11–RAD50–NBS1) complex is required for the activation of ataxia telangiectasia mutated (ATM) in response to double-strand breaks (DSBs). On the other hand the ATM- and Rad3-related (ATR)-interacting protein (ATRIP) complex is recruited to sites of single-strand breaks and activates ATR. Specifically, the CHK2 pathway is activated by DSBs that occur either directly by exposure to ionizing radiation (and radiomimetic agents) or indirectly by topoisomerase II inhibitors. It can also be activated by replication-mediated DSBs as a result of base-pair excision generated by alkylating agents or single-strand breaks caused by topoisomerase I inhibition. Depending on the type of stress, activated CHK1 and CHK2 can phosphorylate a number of overlapping or distinct downstream effectors, which results in the activation of DNA repair, cell-cycle arrest, senescence or apoptosis. Symbols in green are CHK2-specific substrates, symbols in blue are CHK1-specific substrates and symbols in red are shared substrates. Downstream CHK1-specific substrates are not represented here.

Figure 4. G2/M checkpoint control Figure 4. G2/M checkpoint control. DNA damage induces the ATM-mediated phosphorylation of chk2 as well as phosphorylation of chk1, both of which phosphorylate cdc25C, promoting its interaction with 14-3-3 proteins and its cytoplasmic sequestration. As a consequence, cdc25C cannot dephosphorylate cdc2, which remains in an inactive state, resulting in G2 arrest. p53 is also activated following DNA damage, inducing both 14-3-3 and p21Waf1/Cip1, both of which are important in the maintenance of cdc2 inhibition and G2 arrest. In cells lacking p53, disruption of cdc25C cytoplasmic sequestration facilitates mitotic entrance of damaged cells, resulting in cell death. Therefore, caffeine, which inhibits ATM-mediated kinase activity, and UCN-01, which targets chk1, limit phosphorylation of cdc25C, allowing activation of cdc2 and mitotic entry and selectively sensitizing p53-deficient cells to DNA-damaging agents. ATM contributes to p53 phosphorylation following DNA damage. This phosphorylation event is inhibited by high doses of caffeine, perhaps explaining why, at high caffeine concentration, some sensitization to DNA damage is also observed in cells expressing wild-type p53, although effects are still more pronounced in cells lacking p53. Low doses of caffeine specifically sensitize p53-deficient cells.

Figure 1. Progression through the stages of the cell cycle is regulated by cyclin-dependent kinases (cdks). Cdks are positively regulated by cyclins, levels of which fluctuate throughout the cell cycle, and negatively regulated by the endogenous cdk inhibitors of the INK4a and Cip/Kip families. Arrows indicate sites of action of flavopiridol and UCN-01. (A) Depiction of cell cycle control and phases of the cell cycle where flavopiridol or UCN-01 act. (B) Mechanism by which flavopiridol blocks cell cycle progression. (C) Abrogation of G2 checkpoint by UCN-01. Following DNA damage, the G2 checkpoint is activated, which allows the cell to remain in G2 until all DNA damage is repaired and, thus, to enter M phase with "intact" DNA. However, UCN-01 treatment of DNA-damaged cells abrogates the G2 checkpoint (IC50 ~50 nM), which allows the cells to progress into M prior to completion of DNA repair, leading to apoptosis. The UCN-01 G2 checkpoint abrogation was found to involve Cdc25C, the cdk1 (Cdc2)-activating phosphatase, which is modulated by chk1, a protein kinase directly affected by UCN-01.

MECCANISMI D’AZIONE DI UCN-01 Inibizione isoforme Ca2+-dipendenti della PKC (IC50 = 30 nM) Inibizione isoforme Ca2+-indipendenti della PKC (IC50 ~ 600 nM) Arresto del ciclo cellulare inibizione CDKs aumento della sintesi di p27kip1 e p21waf1 Abolizione del checkpoint in G2

EFFETTI TOSSICI DI UCN-01 Nausea/vomito Iperglicemia Tossicità polmonare

Cell division cycle 25 (CDC25) proteins reported to be overexpressed in primary tumour samples from patients with breast96, prostate84, ovarian163, endometrial104, colorectal88, 89, oesophageal91, 92, 94, 112, thyroid96, 97, 98, laryngeal99, gastric101 and hepatocellular cancers100, glioma107, neuroblastoma106 or non-Hodgkin lymphoma109. Percentages of tumours in which CDC25A, CDC25B or CDC25C proteins are overexpressed are indicated, or in the case of overexpression reported in more than one study, a range of percentages is given. The cut-off mark for inclusion was overexpression in >10% patients. Whether (Y) or not (N) CDC25 overexpression was linked to clinicopathological features, including tumour grade or stage, metastases, depth of invasion, residual or recurrent disease, or disease-free survival is marked in yellow boxes beside percentages. Cases for which several studies reported contradictory prognostic values are marked by a (C), and studies in which clinicopathological features were not assessed are indicated by an (?).

. b | Although initial studies suggested a specific role for each CDC25 phosphatase at defined stages of the cell cycle, the current model is that CDC25A, B and C are all involved in phosphorylating CDK–cyclin complexes, such as CDK2–cyclin E at the G1–S transition or CDK1–cyclin B at the entry into mitosis.

c | CDC25A, B and C control entry and progression into mitosis c | CDC25A, B and C control entry and progression into mitosis. CDC25B is thought to be responsible for the initial activation of CDK1–cyclin B at the centrosome that contributes to microtubule network reorganization and mitotic spindle assembly. Nuclear translocation leads to an auto-amplification process (bold arrows) of CDC25s that then fire the bulk of CDK1–cyclin B complexes and trigger mitosis.

The checkpoint hurdle to enter mitosis. a, b | Upon DNA damage, cell division cycle 25 (CDC25) proteins are inhibited through various mechanisms, including checkpoint kinase-dependent degradation or cytoplasmic sequestration through 14-3-3 binding.The inhibitory kinases WEE1 and MYT1 are activated by checkpoint kinases. CDK–cyclin complexes are in turn maintained in their inactive state and the cell remains arrested in G2 phase. c | CDC25B level is central to the control of entry into mitosis after DNA damage. Together with PLK1 activity (which is required for WEE1 inhibition), CDC25B accumulation through a mechanism that is still unclear is required to allow entry into mitosis when damage has been repaired. Increased levels of CDC25B protein found in cancer might therefore facilitate checkpoint exit and increase genomic instability-.

Figure 1 | Structure of human Aurora kinases Figure 1 | Structure of human Aurora kinases.   The domain structure of Auroras A, B and C, along with their size, is shown. The catalytic domain of Auroras A, B and C is highly conserved (green region). Autophosphorylation of Thr288 in the activation loop of Aurora-A is required for the activation of its kinase activity. A short amino-acid peptide motif called the 'destruction box' (D-box) is present in the carboxy-terminal region of Auroras A, B and C. The D-box is recognized by adaptors of the anaphase-promoting complex/cyclosome and thereby targets these proteins for degradation through the ubiquitin/proteasome-dependent pathway. Slide show: presents all available images in this article Nature Reviews Cancer 5, 42-50 (2005); doi:10.1038/nrc1526 AURORA-A — A GUARDIAN OF POLES

Figure 2 | Aurora-A and -B localize to key mitotic structures Figure 2 | Aurora-A and -B localize to key mitotic structures.   Subcellular localization of Aurora-A (red) and Aurora-B (green) relative to the chromosomes (blue) during (a) prophase, (b) prometaphase, (c) metaphase, (d) anaphase and (e) telophase. During prophase, Aurora-A localizes to the centrosomes, whereas in later stages of mitosis it is at the spindle poles (c, d and e) and also extends up the spindle. During prometaphase and metaphase (a and b), Aurora-B localizes to the centromeres. After anaphase (d), however, Aurora-B localizes to the spindle midzone, and finally accumulates at the midbody during telophase (e). Slide show: presents all available images in this article Nature Reviews Cancer 4, 927-936 (2004); doi:10.1038/nrc1502 AURORA-KINASE INHIBITORS AS ANTICANCER AGENTS

Overview of the different effects that are observed on overexpression and/or amplification of AURKA.

Schematic representation of AURKA interactions. Schematic representation of AURKA interactions. AURKA overexpression inhibits p53 family members and suppresses apoptosis and cell cycle arrest. AURKA interacts directly with p53 by phosphorylating it at Ser215 and 315 causing its degradation through MDM2 or inactivating it at transcription level, respectively. AURKA regulates p73 and its downstream targets . It also upregulates the PI3 kinase pathway that enhances cell survival and proliferation either directly interacting with GSK-3β or by regulating AKT (36, 75).

Figure 2 | Localization of Aurora-A and -B kinases during the cell cycle. Aurora-A (blue circles) is first detected at the centrosome during late S phase. The activation of a small proportion of Aurora-A at centrosomes was first evident before chromatin condensation at late G2 phas. Both the amount and activity of Aurora-A rapidly increase in the centrosome, and a fraction of active Aurora-A subsequently translocates into nucleus coincident with chromatin condensation at prophase. After nuclear-envelope breakdown (NEBD), activated Aurora-A is observed at the spindle poles and bipolar spindles during prometaphase and metaphase. The amount of Aurora-A starts to decrease at the metaphase–anaphase transition, but a small fraction of Aurora-A remains on the centrosomes and the spindles at the onset of anaphase and localizes on the spindle midzone and centrosomes during late anaphase and telophase. Aurora-A is degraded by the Cdh1/Fizzy-related form of the anaphase-promoting complex/cyclosome (APC/C). At the final stage of cytokinesis, most of the Aurora-A protein becomes undetectable. Aurora-B (green squares) is a chromosome passenger protein, localizing on chromosome arms during prophase and the interface between sister centromeres (inner centromere region) during prometaphase and metaphase. It then relocalizes to the central spindle and the cell cortex, at the site of cleavage-furrow ingression in the late phases of mitosis. During cytokinesis, Aurora-B localizes at midbody. Recently, it has been shown that Aurora-C is also a chromosome passenger protein that colocalizes with Aurora-B.

Aurora-A activation. Formation of the bipolar spindle requires activation of Aurora-A by the targeting protein for XKLP2 (TPX2). TPX2, which exists in an inhibitory complex with importin-α/ß at the onset of mitosis, is released by Ran-GTP and is then free to bind to Aurora-A. TPX2 interferes with the inhibitory activity of protein phosphatase 1γ (PP1γ) upon Aurora-A and enables Aurora-A to autophosphorylate, thereby activating itself and other substrates, including TPX2. Activated Aurora-A then recruits spindle assembly factors, such as Eg5, that are necessary for the formation of the bipolar spindle. Carvajal, R. D. et al. Clin Cancer Res 2006;12:6869-6875

Figure 3 | Molecular interactions required for centrosome maturation Figure 3 | Molecular interactions required for centrosome maturation.   a | Aurora-A and centrosomin accumulate at the centrosome. b | TACC (transforming acidic coiled coil) is then phosphorylated by Aurora-A, leading to the complex formation with XMAP215 (c). XMAP215 promotes the rapid assembly of microtubules. Microtubules are organized from the centrosome/microtubule-organizing centre, which is composed of a pair of centrioles and the surrounding pericentriolar material (PCM). At the onset of M phase, various proteins including -tubulin, kinases, motor proteins and other pericentriolar material components are recruited to PCM, which changes microtubule nucleating capacity. This remodelling of the PCM is referred to as 'centrosome maturation'.

Figure 4 | Diagram depicting the predicted tumorigenesis by Aurora-A overexpression.   Increases in Aurora-A levels, through gene amplification, transcriptional upregulation or protein stabilization, induces abnormal spindle formation and cytokinesis failure. In normal cells, spindle defects would activate the spindle checkpoint and the cells would undergo arrest at metaphase, but Aurora-A overexpressing cells bypass this checkpoint and instead undergo cytokinesis failure, resulting in formation of tetraploid cells. In cells with intact p53–RB signalling, tetraploidy activates the postmitotic G1 checkpoint, leading to apoptosis or senescence. In cells with impairments in the p53–RB pathway, tetraploid cells tend to have centrosome amplification, which induces chromosome instability. Furthermore, the DNA-damage-induced G2 checkpoint is impaired in Aurora-A overexpressing cells, which also contributes to genomic instability. Overexpression of mutated forms of Aurora-A and the phosphorylation of inappropriate substrates are speculative pathways (dashed lines) by which Aurora-A might also induce tumorigenesis.

Aurora-B and the establishment of chromosome biorientation Aurora-B and the establishment of chromosome biorientation. A, in most cases, the initial kinetochore-microtubule attachment is a monotelic attachment where only one kinetochore (black) is attached to one pole of the spindle (dotted circle). A second kinetochore-microtubule attachment then occurs between the sister chromatid and the opposite spindle pole, thus producing a correct amphitelic attachment state. B, incorrect attachments can occur if both kinetochores establish microtubule connections to the same pole (syntelic attachment) or if one kinetochore is attached to both poles (merotelic attachment). Aurora-B senses the unequal tension across the kinetochore pairs caused by these aberrant attachment modes and decreases the stability of the syntelic and merotelic attachments. In this manner, the generation of the amphitelic attachment state necessary for proper chromosome segregation is promoted.

Figure 3 | Accurate chromosome segregation requires chromosome bi-orientation.   Schematic representation of a metaphase spindle with the centrosomes/spindle poles (red), the chromosomes (blue) and kinetochores (green). Spindle and astral microtubules are represented by thin black lines, whereas kinetochore fibres, which contain about 10 microtubules, are shown as thicker black lines. One correctly bi-oriented chromosome (lower centre), with the sister kinetochores attached to opposite poles, is shown, along with three mal-oriented chromosomes (upper, lower left, lower right). Monotelic/mono-oriented means that only one kinetochore is attached to one pole; merotelic means that one kinetochore is attached to both poles; amphitelic/bi-oriented means that kinetochores are attached to opposite poles; and syntelic means that both kinetochores are attached to the same pole.

Figure 4 | Aurora-B resolves syntelic orientations Figure 4 | Aurora-B resolves syntelic orientations.   Image of a metaphase cell stained to detect microtubules (green), kinetochores (blue) and Aurora-B (red), which localizes to the centromere region between the two sister kinetochores (see enlargement). The schematics show that bi-orientation (lower drawing) results in tension across the centromere, increasing the inter-kinetochore distance. Current models indicate that in the absence of tension (upper drawing), Aurora-B is active at the centromere, which destabilizes syntelic attachments and activates the spindle checkpoint. Following bi-orientation, tension ensues, inactivating Aurora-B and therefore stabilizing bound microtubules as well as silencing the checkpoint. In the presence of Aurora-kinase inhibitors, the lack of tension is not detected — mal-orientations are therefore not resolved and the checkpoint is not activated. Drug-treated cells then exit mitosis prematurely without correctly segregating their chromosomes, yielding tetraploid cells with excess centrosomes. In the absence of the p53-dependent post-mitotic checkpoint, these aberrant cells continue to divide, which ultimately results in cell death.

Pharmacological versus genetic inhibition  Why does treatment with small-molecule inhibitors of Aurora-kinase activity result in less marked phenotypes than other approaches to blocking enzyme function? Although there is no clear explanation, it is not surprising that different methods of inhibiting any given kinase yield different effects — especially if that kinase is part of a multiprotein complex that has structural as well as catalytic roles. Indeed, there is a fundamental difference between selectively inhibiting the enzymatic activity of a kinase versus depleting the protein from a cell. When a kinase is directly inhibited with a small molecule, the stoichiometry of the kinase with respect to binding partners is likely to be unaffected. By contrast, blocking expression through RNA interference (RNAi) or expressing a dominant-negative form of a protein will, by definition, affect the stoichiometry and might disrupt the normal localization and function of binding partners. The figure shows that in normal cells (a) the complex between Aurora-B, inner centromere protein and survivin (ArB–INCENP–Srv) localizes to centromeres and is active, resulting in the appropriate phosphorylation (P) of substrates (S). When cells are exposed to small-molecule inhibitors of kinase activity such as ZM447439, the complex localizes to centromeres, but substrates are not phosphorylated (b). When Aurora-B is repressed by RNAi (c), or a kinase-dead, dominant-negative mutant (K106R) overexpressed (d), substrates are also not appropriately phosphorylated. However, the altered stoichiometry of Aurora-B relative to INCENP and survivin results in disruption of this protein complex, which could have other effects in addition to simply reducing kinase activity.

Aurora kinase inhibitors

Figure 2: Localization and function of Survivin (and its fellow passenger proteins) during cell division. (a) In (pro)metaphase, Survivin localizes on centromeres and chromosome arms (not visible). During anaphase, Survivin no longer associates with centromeric DNA but binds to the overlapping bundles of antiparallel microtubules of the central spindle that form the midzone. These microtubules bundles become compact and mature into the midbody during telophase and cytokinesis, where Survivin eventually localizes. (b) When localized on centromeres, the CPC, of which Aurora B is the enzymatic core and Survivin an important targeting subunit, executes different functions that are important for proper chromosome alignment and segregation: (i) destabilization of improper kinetochore–microtubule attachments and thereby promoting biorientation; (ii) regulation of BubR1 kinetochore levels; (iii) keeping the spindle checkpoint active in the absence of tension by recreating unattached kinetochores and/or by regulating BubR1 kinetochore levels; and (iv) stabilizing chromosome/kinetochore-induced microtubule formation. (v) When present on the central spindle and midbody, the complex is essential for completion of cytokinesis by phosphorylating several substrates (e.g. MKLP1 and vimentin) involved in midzone and midbody function. Abbreviation: MT, microtubule.

Figure 2: Localization and function of Survivin (and its fellow passenger proteins) during cell division. (b) When localized on centromeres, the CPC, of which Aurora B is the enzymatic core and Survivin an important targeting subunit, executes different functions that are important for proper chromosome alignment and segregation: (i) destabilization of improper kinetochore–microtubule attachments and thereby promoting biorientation; (ii) regulation of BubR1 kinetochore levels; (iii) keeping the spindle checkpoint active in the absence of tension by recreating unattached kinetochores and/or by regulating BubR1 kinetochore levels; and (iv) stabilizing chromosome/kinetochore-induced microtubule formation. (v) When present on the central spindle and midbody, the complex is essential for completion of cytokinesis by phosphorylating several substrates (e.g. MKLP1 and vimentin) involved in midzone and midbody function. Abbreviation: MT, microtubule.