Estudo Dirigido - Ciclo Celular e Apoptose

O início da apoptose ocorre a partir de um sinal apoptótico As caspases são proteases presentes no interior da célula que permanecem inativas e somente são ativadas no momento da apoptose O sinal apoptótico leva a junção de diversas caspases iniciadoras em grandes complexos e dentro desses complexos as capazes iram formar dímeros A formação do dímero torna a caspase ativada A partir de sua ativação em cada dímero uma caspase cliva seu par em um determinado sitio adequando o funcionamento da enzima Os dímeros de caspases iniciadoras ativadas e clivadas iram ativar as caspases executoras que se encontram no interior da célula na forma de dímero inativos A ação da caspase incitadora sobre a caspase executora leva a alteração da sua forma e torna a mesma ativa A caspase executora agora ativa atua como catalisador no processo de clivagem de diversas proteínas intracelulares como lâmina nucleares componentes do citoesqueleto e proteínas de adesão intercelular Outra ação da caspase executora é a liberação da endonuclease que normalmente se encontra ligada a uma proteína A endonuclease livre fragmenta o DNA celular presente no núcleo O sinal apoptótico é debelado pela ligação de proteínas sinalizadoras extracelulares que se ligam a receptores na superfície da célula e ativam a via extrínseca da apoptose A ligação a proteína de membrana leva a ligação de proteínas intracelulares que ativam as caspases iniciadoras No caso da via apoptótica intrínseca a liberação de proteínas mitocondriais para o citoplasma celular leva a ativação de caspases citoplasmáticas que conduzem a célula a apoptose Um exemplo de proteína mitocondrial que quando presente no citoplasma leva a apoptose é a proteína citocromo c que quando liberada se liga ao fator 1 de ativação da protease apoptótica Apaf1 A Apaf1 se liga a outras Apaf1 formando um complexo cíclico composto por 7 cadeias peptídicas que recebe o nome de apoptossomo O apoptossomo recrutará a caspase iniciadora que quando ativada ativa a caspase executora que atuará como catalisador de diversas clivagens proteínas no interior celular Durante a apoptose em decorrência dos eventos celulares a células serão condensadas com ocorrência de colapso do citoesqueleto degradação o envelope nuclear além de condensação e fragmentação da cromatina No caso de células grandes pode ocorrer formação de corpos apoptóticos que são fragmentos celulares da célula em processo de morte por apoptose contidos em uma membrana esses corpos apoptóticos ou a célula em apoptose inteira são rapidamente fagocitados por macrófagos ou englobadas pelas células vizinhas As células mortas por necrose no entanto explodem sobre as células adjacentes Tal ocorre por alteração no gradiente iônico em decorrência de depleção energética ou outros fatores A célula que morre por necrose irá sofrer um processo de expansão até que haja rompimento de sua membrana e liberação do conteúdo nas células vizinhas A imagem C corresponde a célula tratada com a droga 2 que induz apoptose A imagem A corresponde a célula controle não tratada A imagem B corresponde a célula tratada com a droga 1 que induz morte celular por necrose UNIVERSIDADE DO ESTADO DO RIO DE JANEIRO INSTITUTO DE BIOLOGIA Roberto Alcantara Gomes DEPARTAMENTO DE BIOLOGIA CELULAR Disciplina de Biologia Celular Estudo Dirigido Ciclo Celular e Apoptose 1 O complexo ciclinaCDK da fase M é regulado de maneira específica diferindo da regulação do complexo ciclinaCDK das outras fases Com base no esquema abaixo descreva o mecanismo de regulação do complexo ciclinaCDK da fase M com ênfase na sua ativação inibição e degradação 2 A transição entre as diferentes fases do ciclo celular é controlada pelos pontos de checagem Neles a célula verifica se o ambiente está favorável se já cresceu o suficiente e se há danos no DNA Diga como é regulado o ponto de checagem em G1 explicando o que são CKis e como elas agem 3 Os eventos celulares podem ser regulados em níveis variados Por exemplo quando uma determinada proteína se torna desnecessária ou mesmo prejudicial à célula uma série de mecanismos de controle são acionados a fim de que os efeitos biológicos desta proteína cessem no interior da célula Assim a síntese da proteína é diminuída inibindose a transcrição do gene que a codifica a tradução de seu RNAm e mesmo a meiavida deste é diminuída Mesmo após estas providências restam no ambiente celular cópias da proteína que precisam ser inativadas inibidas ou destruídas As proteínas que atuam no controle do ciclo celular também estão submetidas a esta lógica vital A partir do esquema abaixo explique como ocorre a regulação da degradação de determinadas proteínas CKI e ciclinas da fase G1 G1S e S durante o ciclo celular e explique porque estas moléculas precisam ser degradadas em determinado momento Pi fosfato inibitório Pa fosfato ativador Ubn ubiquitina Degradação no proteossomo Fase G1S 1 4 Atualmente se sabe que a proteína p53 tem um duplo papel no controle do ciclo celular Inicialmente como um fator de transcrição aumentando a expressão de um tipo de CKI a proteína p21 Entretanto a p53 também pode influenciar o processo de liberação de citocromo c para o citosol por mecanismos que ainda estão sendo estudados Com base nestas informações explique que via de apoptose pode ser induzida pela P53 descrevendo as moléculas envolvidas e o seu papel na apoptose 5 Um pesquisador estava estudando duas drogas distintas 1 e 2 e observou os efeitos desta droga na morfologia de uma determinada linhagem de células epiteliais Ele incubou as células durante a noite na presença das duas drogas No dia seguinte o pesquisador processou as células para realizar uma microscopia eletrônica O pesquisador observou que uma das drogas induzia a célula a morrer por necrose enquanto a outra por apoptose Em seguida ele tirou algumas algumas fotos para exemplificar as mudanças morfológicas ocorridas no controle não tratadas e nas células que ficaram na presença da droga 1 e da droga 2 Analise as figuras abaixo e indique que foto pertence as células controle e as células tratadas com a droga 1 ou 2 explicando porque você fez esta escolha Quais alterações morfológicas são específicas para cada tipo de morte celular e que eventos bioquímicos levam a estas alterações morfológicas INVITED REVIEW Journal of Pathology J Pathol 2012 226 352364 Published online 28 October 2011 in Wiley Online Library wileyonlinelibrarycom DOI 101002path3022 The cell cycle and cancer Gareth H Williams12 and Kai Stoeber12 1 Department of Pathology and Cancer Institute University College London UK 2 Wolfson Institute for Biomedical Research University College London UK Correspondence to Gareth H Williams Department of Pathology University College London Rockefeller Building 21 University Street London WC1E 6JJ UK email garethwilliamsuclacuk Abstract Deregulation of the cell cycle underlies the aberrant cell proliferation that characterizes cancer and loss of cell cycle checkpoint control promotes genetic instability During the past two decades cancer genetics has shown that hyperactivating mutations in growth signalling networks coupled to loss of function of tumour suppressor proteins drives oncogenic proliferation Gene expression profiling of these complex and redundant mitogenic pathways to identify prognostic and predictive signatures and their therapeutic targeting has however proved challenging The cell cycle machinery which acts as an integration point for information transduced through upstream signalling networks represents an alternative target for diagnostic and therapeutic interventions Analysis of the DNA replication initiation machinery and mitotic engine proteins in human tissues is now leading to the identification of novel biomarkers for cancer detection and prognostication and is providing target validation for cell cycledirected therapies Copyright 2011 Pathological Society of Great Britain and Ireland Published by John Wiley Sons Ltd Keywords biomarker cell cycle cancer DNA replication MCM geminin phosphohistone mitosis therapy Received 17 August 2011 Revised 30 September 2011 Accepted 1 October 2011 No conflicts of interest were declared Introduction The majority of cells in the human body are not cycling and instead reside in outofcycle states A minority of cells are actively cycling proliferating and these are located mainly in the stemtransit amplifying com partments of selfrenewing tissues such as epithelia and bone marrow 1 In contrast most functional cells have irreversibly withdrawn from the cell division cycle into terminally differentiated states eg neurones myocytes or surface epithelial cells of skinmucosa or have reversibly withdrawn into a quiescent G0 state eg glial cells thyroid follicular cells or hepatocytes 23 The cell cycle has four sequential phases Arguably the most important phases are S phase when DNA replication occurs and M phase when the cell divides into two daughter cells Separating S and M phase are two gap phases referred to as G1 and G2 G1 follows on from mitosis and is a time when the cell is sensitive to positive and negative cues from growth signalling networks G2 is the gap after S phase when the cell prepares for entry into mitosis 4 G0 represents a state when cells have reversibly withdrawn from the cell division cycle in response to high cell density or mitogen deprivation 5 Alter natively cells may irreversibly withdraw from the cell cycle into terminally differentiated or senescent outofcycle states Progression through the cell cycle is driven by the cyclindependent kinase CDK family of serinethreonine kinases and their regulatory part ners the cyclins 6 Cyclin DCDK4 cyclin DCDK6 and cyclin ECDK2 drive G1 progression through the restriction point which commits the cell to complete the cycle 7 S phase is initiated by cyclin ACDK2 and cyclin BCDK1 regulates progression through G2 and entry into mitosis 8 Progression through each cell cycle phase and transi tion from one phase to the next are monitored by sensor mechanisms called checkpoints which maintain the correct order of events 9 If the sensor mechanisms detect aberrant or incomplete cell cycle events eg DNA damage checkpoint pathways carry the signal to effectors that can trigger cell cycle arrest until the problem is resolved 1011 Effector proteins include the CDK inhibitors CDKIs which can reversibly halt cell cycle progression For example G1 arrest can be induced through the action of the Ink4 family INK4A p16 INK4B p15 INK4C p18 and INK4D p19 of CDKIs which inhibit CDK4 and CDK6 or alterna tively via the CipKip family of inhibitors p21 p27 p57 which suppress CDK2 activity 1213 Deregulation of the cell cycle engine underlies the uncontrolled cell proliferation that characterizes the malignant phenotype Mitogens release the brakes of cell cycle progression by stimulating G1S CDK activities which trigger the phosphorylation of pRB proteins leading to disruption of their interaction with Copyright 2011 Pathological Society of Great Britain and Ireland J Pathol 2012 226 352364 Published by John Wiley Sons Ltd wwwpathsocorguk wwwthejournalofpathologycom The cell cycle and cancer 353 the E2F family of transcription factors In cancer cells the pRB brakes are often defective resulting in E2F dependent G1S gene expression even in the absence of mitogens 14 This may arise as a result of acti vating tumourigenic mutations which have been iden tified in diverse tumours at all levels in the mitogenic signalling pathways from ligands and receptors eg HER2ErbB2neu receptor mutations or HER2 gene amplification to downstream signalling networks eg RasRafMAPK or PI3KAkt signalling pathways and also for the cell cycleregulated genes them selves eg CYCLIND1 and CDK4 gene amplification 1517 Aberrant signalling promotes activation of CDKcyclin complexes which phosphorylate Rb and attenuate its capacity to induce transcriptional repres sion The notion that Rb phosphorylation is a conver gence point for these oncogenic signalling pathways is consistent with the fact that inactivation of the RB gene by mutation or methylation is a common occur rence in cancer 18 The inactivation of tumour sup pressor genes that encode CDKIs eg p15 p16 and p27 are also common events in diverse tumour types This releases the brakes on cell cycle progression and further abrogation of checkpoint control mechanisms leads to the acquisition of genomic instability which drives tumour evolution 12 The cell cycle machinerya convergence point for oncogenic signalling pathways Analysis of the complex and partly redundant upstream signalling networks that control processes such as cell proliferation differentiation and invasion by genome wide analysis remains to be proven as a routine tool for clinicopathological assessment The early studies using microarraybased gene expression profiling led to the identification of potentially powerful prognostic and predictive signatures suggesting that this technol ogy might soon replace traditional clinicopathologi cal parameters 1921 However subsequent studies have shown that the prognostic and predictive power of microarrays provides only complementary informa tion and cannot be used as a replacement for tra ditional clinicopathological variables Disappointingly the actual performance of prediction rules using gene expression has not been as informative as hoped for many tumour types and the list of genes identified can be highly unstable 2223 For instance assign ment of molecular subtype classes of breast can cer based on the analysis of dendrograms obtained with hierarchical cluster analysis has proven subjec tive with modest interobserver reproducibility 24 Whether prognostic signatures will reduce the num ber of patients undergoing toxic chemotherapy remains unclear For example although the MammaPrint 70 gene signature is expected to identify 1015 of patients who might be spared chemotherapy results of a recent finalized feasibility study suggest this outcome is overly optimistic 25 Transcriptomic profiling has also proved constrained as a predictor of therapeu tic response Predictive signatures do not consistently correlate with treatment response and their predictive value has been significantly reduced when applied to validation cohorts 2627 Moreover retrieval of sur gical material for microarray analysis particularly for small tumours presents a formidable challenge in the routine clinical setting together with associated cost implications The complex nature of these signalling networks also compromises the approach of using targeted therapies The oncogene addiction theory suggests a tumour will have unyielding dependence on a particular perturba tion of a single gene 28 The reality is that cancer cells are unstable and have many alterations 29 Hence the cancer circumnavigates the specific target and eludes the targeted therapy 1530 This phenomenon is reflected in the largely disappointing results of tar geted therapy clinical trials in cancer where at best there is a change to the natural history of the tumour but there is still no cure 3138 An alternative approach is to focus on the cell cycle machinery which acts as an integration point for information transduced through upstream signalling networks 33940 Notably many of the current most effective neoadjuvant and adjuvant therapeutic interventions in the clinic are cell cycle directed agents Table 1 A core component of the cell division cycle the DNA replication initiation pathway has emerged as a target of particular interest over the last decade 34143 The DNA replication initiation machinery can be regarded as a final and critical step in growth control positioned at the convergence point of complex and branched upstream signalling networks 40 This component of the cell cycle engine acts as a relay station connecting growth signalling networks with the initiation of DNA synthesis and is therefore a potentially attractive diagnostic and therapeutic target 3 Here we review the recent literature on cell cycle proteins as cancer biomarkers with particular empha sis on DNA replication initiation factors and mitotic engine proteins We also discuss the emerging concept of targeting the replication initiation machinery for can cer therapy We apologize to the many authors whose important contributions we could not cite due to space limitations The DNA replication initiation pathway DNA synthesis is tightly controlled to ensure that replication origins are not fired more than once per cell cycle 44 This is achieved through a replica tion licensing system that coordinates DNA replica tion initiation events at chromosomal origins with cell cycle progression The licensing machinery is com posed of a complex of initiator proteins that bind Copyright 2011 Pathological Society of Great Britain and Ireland J Pathol 2012 226 352364 Published by John Wiley Sons Ltd wwwpathsocorguk wwwthejournalofpathologycom 354 GH Williams and K Stoeber Table 1 Cell cycletargeted therapeutic agents Agent Class Target Phase affected 5Fluorouracil Antimetabolite Thymidylate synthase S Gemcitabine Antimetabolite Nucleoside analogue and ribonucleotide reductase S Methotrexate Antimetabolite Dihydrofolate reductase S Irinotecan Camptothecin Topoisomerase I S Cisplatin Alkylating agent DNA interstand crosslinks SG2 Docetaxel Taxane Tubulin M Paclitaxel Taxane Tubulin M Vincristine Vinca alkaloids Tubulin M to and unwind the DNA helix at origins prior to the formation of bidirectional replication forks During late M and early G1 phase the licensing proteins ORC Cdc6 Cdt1 and Mcm27 assemble into pre replicative complexes preRCs thereby rendering ori gins licensed for DNA synthesis during S phase 4546 The six MCM proteins Mcm27 function as a replicative helicase unwinding the template DNA with Cdc6 and Cdt1 acting as clamploaders for this ringshaped heterohexameric complex 4749 At the transition from G1 into S phase licensed replication origins are fired by the concerted action of CDKs and the Dbf4dependent Cdc7 kinase 50 Cdc7 phospho rylates the Mcm2 4 and 6 subunits thereby inducing a conformational change that stimulates MCM helicase activity 5153 The formation of an active helicase leads to the recruitment of additional factors including Cdc45 and the four subunit GINS complex which is also dependent on Cdc7 kinase activity 5456 Once activated the MCM helicase unwinds doublestranded DNA at origins to generate a singlestranded DNA template required to recruit the DNA synthesis machin ery containing RPA PCNA and DNA polymerase αprimase 46 Following entry into S phase the licensing system is shut down to prevent reinitiation events at origins that have already been fired The key event in suppressing relicensing of origins is the inactivation of the MCM loading factor Cdt1 through two mechanisms 57 First Cdt1 undergoes cell cycle dependent proteolysis during S and G2 58 Sec ond residual Cdt1 is inhibited by the binding of a small regulatory protein called geminin which is expressed at high levels during the S G2 and M phases 5961 Defining the proliferative state Investigation of the DNA replication initiation machin ery in different organisms tissues and cell types has revealed that cell cycle withdrawal and loss of proliferative capacity are linked to a shutdown of the licensing system 3436264 During the proliferationdifferentiation switch the MCM clamp loaders Cdc6 and Cdt1 are rapidly downregulated as cells migrate from the transit amplifying com partment to the functionally differentiated compart ment of selfrenewing tissues There is a more gradual downregulation of Mcm27 proteins as cells mature and adopt a fully differentiated phenotype The conversion of replication origins into an unli censed state also characterizes the quiescent G0 and senescent outofcycle states and therefore appears to be a common mechanism by which proliferation is restrained in multicellular organisms 363 Figure 1A B Interestingly the regulation of Cdc6 protein lev els appears to coordinate the proliferative capacity of cells during cell cycle withdrawal and reentry Its downregulation triggers loss of proliferative capacity during early engagement of the somatic differentiation programme while during cell cycle reentry G0S CDK phosphorylation of Cdc6 prevents its destruc tion by the anaphasepromoting complex APC thus facilitating the licensing of origins 6566 Prolifer ating cells are characterized by high expression lev els of the MCM proteins throughout the cell divi sion cycle with cyclical binding to origins occurring in late Mearly G1 and displacement from chromatin during S phase 6267 Consequently Mcm27 have emerged as novel biomarkers of proliferation Unli censed replication origins and absence of CDK activ ity on the contrary characterize the differentiated and G0 outofcycle states and therefore allow such cells to be clearly distinguished from cycling cells in complex and dynamic heterogeneous cell populations 4362 DNA replication licensing and cancer Identification of MCM proteins in pathological speci mens using immunodetection methods has been shown to be an accurate and simple method for determin ing the growth fraction in dynamic tumour cell pop ulations 396870 Figure 1B Moreover Mcm27 expression levels are powerful prognostic indicators in diverse tumour types including cancers of the lung breast kidney bladder prostate and ovary 7177 This finding is consistent with largescale metaanalysis of cancer microarray data which identi fied upregulation of the MCM26 genes as a com ponent of poor prognostic signatures 78 In most tumour types the upregulation of MCM and other licensing proteins is likely to reflect oncogenedriven engagement of the cell division cycle Indeed many components of the DNA replication initiation machin ery are under E2F transcriptional control eg Cdc6 Copyright 2011 Pathological Society of Great Britain and Ireland J Pathol 2012 226 352364 Published by John Wiley Sons Ltd wwwpathsocorguk wwwthejournalofpathologycom The cell cycle and cancer 355 Figure 1 Expression of DNA replication initiation proteins in selfrenewing tissues A Schematic showing relative protein expression levels for the initiation proteins Cdc6 Cdt1 required for loading the Mcm27 complex onto chromatin and Mcm27 replicative helicase in stem cell transitamplifying and differentiated compartments The schematic drawing of a colonic crypt illustrates this hierarchal organization of selfrenewing tissues The flux of cells through these compartments is continuous new cells are supplied from the stem cell compartment and their number is multiplied in the transitamplifying compartment Cells become functionally competent as they enter the fully differentiated compartment The stem cell compartment is characterized by low expression of initiation proteins Cdc6 Cdt1 and Mcm27 levels rapidly increase as cells enter the transitamplifying compartment There is a gradual downregulation of Mcm27 as cells mature and adopt a fully differentiated functional phenotype Proliferative capacity however is lost at an earlier point in the differentiation programme as cells exit the transitamplifying compartment and is linked to downregulation of the loading factors Cdc6 and Cdt1 626366 Notably the arrested differentiation that characterizes cancer particularly in highgrade tumours is associated with failure to downregulate the replication initiation proteins B Spatial organization of Mcm27 protein expression in normal oesophageal squamous epithelium and nondysplastic Barretts mucosa and disruption of this highly organized spatial arrangement in premalignant dysplasia and invasive cancer 6996 In normal squamous epithelium high expression levels of Mcm2 above a welldefined basal layer fall to undetectable levels in the upper third In squamous epithelial dysplasia Mcm2 expression persists to the luminal surface Invasive squamous cell carcinoma shows high levels of Mcm2 expression Nondysplastic intestinal type Barretts mucosa shows Mcm2 expression in cells of the proliferative zone beneath the mucosal surface Expression falls away markedly on the mucosal surface In Barretts mucosa showing mild dysplasia Mcm2 downregulation does not occur Invasive adenocarcinoma shows high levels of Mcm2 expression Cdt1 Mcm27 Mcm10 and Dbf4 7983 How ever deregulation of the licensing system may also be a primary driver of oncogenesis at least in some tumour types For example overexpression of Cdc6 or Cdt1 have been shown to be oncogenic and deregulated Mcm7 expression has been linked to tumour formation progression and malignant transfor mation in animal models 8490 Oncogenic muta tions in genes upstream of the licensing machinery eg RAS CYCLINE and CYCLIND1 can also impact on tumourigenesis by causing deregulation of the licens ing machinery This may either result in relicensing events or allow cells to enter S phase with insufficient licensed origins see below both of which can lead to genomic instability 42 Replication initiation proteins in cancer detection and screening In normal selfrenewing epithelia eg found in blad der cervix skin gut and the airways movement of cells from the stemtransit amplifying to the functionally differentiated compartment is coupled to a shutdown of the licensing system and loss of pro liferative capacity 343 Thus licensing proteins are normally restricted to the proliferative compartment Copyright 2011 Pathological Society of Great Britain and Ireland J Pathol 2012 226 352364 Published by John Wiley Sons Ltd wwwpathsocorguk wwwthejournalofpathologycom 356 GH Williams and K Stoeber and are absent from the functional compartment 63 Figure 1A B The important role that repression of the licensing system plays in proliferation control is highlighted by the finding that Cdc6 overexpression sustains the proliferative capacity of differentiating cells 66 The normal somatic differentiation programme is disrupted in premalignant epithelial lesions referred to pathologically as dysplasia or intraepithelial neoplasia These early noninvasive lesions are characterized by the emergence of cytologically abnormal cells abroga tion of the normal differentiation programme with loss of epithelial polarity and an increase in the size of the proliferative compartment Intriguingly the switch to the dysplastic state is associated with a failure to down regulate the licensing system resulting in high MCM protein expression in all epithelial layers including the surface cells 3396869 This expression pattern indi cates that the majority of cells in premalignant lesions are locked into the cell division cycle Notably only a small proportion of these cells express geminin a marker for cells in S G2 and M phases showing that the majority of neoplastic cells fail to progress through the cell cycle and therefore reside in a G1 extended or arrested state Deregulation of the Mcm27 licensing factors in premalignant and malignant lesions has been exploited in the development of a number of cancerdiagnostic applications The detection of exfoliated MCM posi tive cells in body fluids eg in urine prostatic secre tions stool samples or gastrooesophageal aspirates or active sampling by swabbing or brushing eg cer vical smears or ERCP brushings for pancreaticobil iary tract sampling provides a sensitive and specific method for the detection of premalignant and malig nant lesions in a range of organ systems 399198 For example the immunostaining of cervical Pap smears for Mcm27 has potential to increase both the sensitivity and specificity of this errorprone test 3999101 Clinical trials are ongoing combining MCM biomarkers with liquidbased cervical cytology and automated microscopy platforms eg BD ProEx CFocalPoint GS Imaging System Notably a recent study has shown that primary hrHPV DNAbased screening followed by BD ProEx C triage antibodies to Mcm2 and Topo2A proteins represents the opti mal cervical screening strategy resulting in 55 fewer referrals for colposcopy 93 An alternative method to detect MCMpositive tumour cells in patient sam ples is to use liquidphase assays such as ELISA or DELFIA Clinical studies using this approach have generated encouraging results for diverse tumour types including the screening of urine sediments for detec tion of transitional cell carcinoma of the bladder and prostate cancer and bile aspirates for pancreaticobil iary tract cancer 94959798 An MCMbased cancer test Figure 2 has therefore broad potential clinical utility in cancer detection tumour surveillance pop ulation screening monitoring of therapeutic response and prognostication Tumour cell cycle phase analysis Tumour cell cycle kinetics not only impacts on prog nostic assessment but is also of potential importance for predicting response to cell cycle phase specific agents Prognostic algorithms for many tumour types include a crude measure of their proliferative state often based on mitotic index andor Ki67 count eg Nottingham Prognostic Index for breast cancer Feder ation Nationale des Centres de Lutte le Cancer grading system for soft tissue sarcoma 102103 Notably many of the neoadjuvant and adjuvant chemothera peutic interventions approved for clinical use include agents targeting either replicating cells in S phase or dividing cells M phase and will therefore only be effective against cells progressing through the cell cycle In support of this concept in breast cancer high Ki67 levels appear to predict benefit for an adjuvant taxane regime M phase agent docetaxel compared with nontaxane regimes 104105 However although Ki67 has emerged as a prognostic marker of poten tial interest its introduction into routine clinical prac tice has been compromised by conflicting data from metaanalysis studies 106 Moreover harmonization in methods used to quantify Ki67 levels between labo ratories has also proved to be problematic and reported cutpoints are highly variable Geminin mitotic kinases and phosphohistone H3 can be used to determine cell cycle position The analysis of core constituents of the cell cycle machinery provides an alternative method to assess the proliferative state of dynamic tumour cell popu lations Figure 3A As discussed above expression of the Mcm27 proteins allows tumour cells engaged in the cell division cycle to be clearly distinguished from cells residing in outofcycle states Geminin which prevents relicensing of replication origins after the initiation of DNA synthesis is only present in cells progressing through S G2 and M phases as are the mitotic engine kinases Plk1 Aurora A and Aurora B 74107108 These three kinases control most mitotic events including centrosome maturation and sepa ration chromosome orientation and segregation 8 Notably histone H3 is a substrate for the Aurora kinases and is phosphorylated at serine 10 only dur ing the length of M phase 108109 Phosphohistone H3 H3S10ph is therefore an M phase marker Hence multiparameter analysis of these G1S and G2M reg ulators using immunodetection methods provides a detailed readout of the cell cycle state in complex dynamic tumour cell populations in human tissues 3 Using this approach to study breast cancer a complex and highly heterogeneous tumour type with respect to cancer genetics and clinicopathological parame ters has revealed three discrete cell cycle phenotypes 110111 Figure 3B These include a an outof cycle state composed predominantly of MCMnegative cells b a G1delayedarrested state composed of cells Copyright 2011 Pathological Society of Great Britain and Ireland J Pathol 2012 226 352364 Published by John Wiley Sons Ltd wwwpathsocorguk wwwthejournalofpathologycom The cell cycle and cancer 357 Figure 2 Schematic of the rationale for MCMbased cancer detection tests Mcm27 protein expression in normal epithelium is restricted to the basal stemtransit amplifying compartments and is absent from surface layers as cells adopt a fully differentiated phenotype MCM expression shown in purple Superficial cells obtained either through exfoliation or by surface sampling should therefore be negative for Mcm27 proteins In premalignant dysplastic epithelial lesions and in malignancy there is an expansion of the proliferative compartment coupled to arrested differentiation resulting in the appearance of proliferating MCMpositive cells in superficial layers Immunodetection of Mcm27 protein in exfoliated or surfacesampled cells is thus indicative of an underlying premalignantdysplastic lesion or malignancy The receiver operating characteristics curves show the high sensitivity and specificity of MCMbased tests for detection of oesophageal bladder and prostate cancer 959698 while the detection of premalignant cells in cervical smears is illustrative of the potential for cytologybased testing 3993 with high MCM expression but low SG2M phase marker expression geminin Plk1 Aurora A and low M phase marker expression H3S10ph and c an accelerated cell cycle progression phenotype with cells showing high MCM SG2M and M phase biomarker expression 110111 The cell cycle phenotype is a powerful independent prognosticator in breast cancer and outperforms the gold standard proliferation marker Ki67 In a study of 182 breast cancers the accelerated cell cycle phenotype had a much higher risk of relapse when compared with the outofcycle and G1delayedarrested phenotypes HR 390 p 0001 110111 These early proof ofconcept studies applying the cell cycle phenotype test are consistent with published gene expression pro filing studies showing that conserved tumour expres sion patterns include many proliferationassociated genes and that increased expression of these socalled proliferation signatures is associated with enhanced malignancy 78112113 It will be of major interest to determine how this simple cell cycle biomarker test which is highly suited to routine surgical biopsy mate rial compares with expensive multigene tests such as Oncotype DX 114 Notably we have discovered that these discrete cell cycle phenotypes appear to be com mon to many other tumour types indicating that the cell cycle biomarker test could be used as a prognos ticator for diverse cancer types 115 and unpublished data DNA replication initiation factors appear to hold some advantages over the use of the gold standard marker Ki67 for determining the proliferative state of dynamic cell populations in tissue samples First the variability in cutpoints for Ki67 prognostic scoring partly reflects uncertainty about its function leading to threshold values being determined empirically In contrast the combined use of MCM geminin and H3S10ph biomarkers provides a cell cycle profile based on the wellcharacterized biological function of these proteins during the cell division cycle Second in our proofofprinciple study the cell cycle biomarker algorithm was able to clearly separate breast cancer into three discrete cell cycle phenotypes whereas the Ki67 labelling index did not clearly separate between these cell cycle states 110 Third these cell cycle biomarkers generate robust strong nuclear signals in Copyright 2011 Pathological Society of Great Britain and Ireland J Pathol 2012 226 352364 Published by John Wiley Sons Ltd wwwpathsocorguk wwwthejournalofpathologycom 358 GH Williams and K Stoeber Figure 3 Tumour cell cycle phase analysis A Schematic showing cell cycle phasespecific expression of the biomarkers Mcm2 Plk1 Aurora A geminin and histone H3 phosphorylated on serine 10 H3p in proliferating cells and outofcycle states G0 differentiation B Three distinct cell cycle phenotypes characterized by differential expression of Mcm2 geminin Plk1 Aurora A and H3p were found in breast cancer 110111 and other cancer types 115 and unpublished data Prognosis and prediction of response to treatment can be derived from the distinct immunoexpression profile displayed by each tumour Patients with tumours comprised predominantly of cells with an accelerated cell cycle progression phenotype are more likely to derive benefit from S andor Mphasedirected chemotherapeutic agents formalinfixed surgical biopsy material making them particularly amenable to scoring algorithms developed for digital pathology platforms Cell cycle phase analysis as a predictor of therapeutic response Whether cell cycle biomarker analysis might be used as a predictor of therapeutic response to cell cycle phase specific agents is an interesting question The disap pointing intenttotreat analyses of large convention ally designed trials such as TACT and tAnGo suggests that further improvements in adjuvant treatment will require individualized therapeutic decisions 116117 Cell cycle phase analysis of breast and ovarian cancers has shown that it is tumours displaying the accelerated cell cycle phenotype that are most likely to show a clinically relevant response to S or Mphasedirected agents Table 1 Figure 3B 374110111118 As nonproliferating cells are radiationresistant whereas cycling cells are most sensitive to radiation insult during transit through G2 and M phase tumours displaying the accelerated cell cycle phenotype may also represent those that are most radiationsensitive During the last two decades molecular genetics research has shown that cancer arises as a result of a complex and unique set of mutations that drive oncogenic proliferation Many of these mutations have been identified in growth signalling pathways lead ing to the development of smallmolecule inhibitors SMIs targeting cellsurface receptors and signalling molecules eg EGFR VEGF KRAS BRAF PI3K MEK ERK 1530119121 This is driving the con cept that rather than describing cancers according to their site of origin and clinicopathological parameters tumours might alternatively be classified in terms of the main pathways that drive tumour cell proliferation eg PI3KPTENmTORdriven cancer Wntdriven can cer etc 122124 However molecular tools to mea sure activation of the signalling pathways in tumour Copyright 2011 Pathological Society of Great Britain and Ireland J Pathol 2012 226 352364 Published by John Wiley Sons Ltd wwwpathsocorguk wwwthejournalofpathologycom The cell cycle and cancer 359 Figure 4 Targeting DNA replication before it startsexploitation of the DNA replication origin activation checkpoint for cancer therapy A Model to illustrate the molecular architecture of the origin activation checkpoint 129 CDC7 knockdown by RNAi or alternatively inhibition of Cdc7 kinase activity with small molecule inhibitors SMIs triggers a cellular response that is dependent on three checkpoint axes coordinated through the cell stress transcription factor FoxO3a Stalling of the cell cycle in G1 is initiated by FoxO3a through activation of the ARFHdm2p5321 pathway and upregulation of p15INK4B and p27CDKN1B P53 in turn activates expression of the Wntβcatenin signalling antagonists Dkk3 leading to Myc and cyclin D1 downregulation The resulting loss of CDK activity inactivates the RbE2F pathway and overrides the G1S transcriptional programme A lack of redundancy between the checkpoint axes and reliance on several tumour suppressor proteins commonly inactivated in human tumours provides a mechanistic basis for the cancer cellspecific killing observed with Cdc7 SMIs in preclinical studies see also B The exact molecular mechanism by which blocked origin firing activates FoxO3a remains to be determined B A Colo205 tumour xenograft top right treated with a Cdc7 SMI shows an extensive area of cell death indicative of a strong cytotoxic effect while the control tumour xenograft top left shows a dense and compact architecture with high mitotic activity Mouse colonic mucosa bottom panels shows normal morphology in both control and inhibitortreated mice with no apparent toxic effects biopsy material have been lacking and it is not clear which pathways are driving oncogenic proliferation Importantly all these pathways converge at the level of the cell cycle machinery driving cells through the restriction point in G1 and culminating in activation of the G1S transcriptional programme It is there fore interesting to speculate whether cell cycle phe notype analysis as described above might also be used as a predictor of response to SMIs targeting upstream growth signalling networks that modulate cell cycle progression Tumours comprised predomi nantly of cells displaying cell cycle phenotypes a and b appear not to be receiving sufficient mitogenic signalling to drive them through the cycle and cell division and therefore should be refractory to such inhibitors 110111 In contrast receptorsignalling Copyright 2011 Pathological Society of Great Britain and Ireland J Pathol 2012 226 352364 Published by John Wiley Sons Ltd wwwpathsocorguk wwwthejournalofpathologycom 360 GH Williams and K Stoeber pathways do appear to be exerting an effect on those tumours displaying the accelerated cell cycle pheno type c although in a proportion of cases this may be the result of the development of autonomous cancer cell cycles following mutations in critical regulators of the cell cycle machinery itself In any case it is only tumours displaying the accelerated cell cycle pheno type c that are likely to show significant response to upstream growth signalling inhibitors now entering the clinic 110111 Cell cycle phase analysis therefore has potential to stratify patients likely to respond to such inhibitors in clinical trials 3110111 Importantly following this rationale cell cycle biomarker analysis might be used to salvage the many reported disappoint ing intenttotreat trials using SMIs 3538 Retro spective cell cycle phase analysis of surgical biopsy material linked to patients participating in these tri als could be performed through utilization of hospital tissue archives The DNA replication initiation machinerya promising anticancer target The targeting of upstream growth signalling pathways is often constrained by pathway redundancy 40 or the development of growthindependent autonomous cancer cell cycles 3 Efficacy can be compromised through a variety of mechanisms eg through over expression of alternative receptor tyrosine kinases or development of new signalling pathways 30 Ther apeutic targeting of the DNA replication initiation machinery which lies at the convergence point of growth signalling networks is now emerging as a new concept promising to overcome the limitation of targeting more upstream pathways A concern is that therapeutic intervention at this level will not dis criminate between rapidly dividing normal cells and tumour cells thus leading to severe systemic side effects while attempting to reduce tumour mass This would translate into low therapeutic indices often found for conventional chemotherapeutic drugs target ing the cell cycle However potent cancer cellspecific killing has been demonstrated in preclinical models after inhibition of origin licensing 125 or alterna tively origin activation through targeting Cdc7 kinase 111118126127 Tumour cell specificity is thought to result from transformed cells entering S phase with inadequate numbers of competent origins to complete chromosomal replication This results in an abortive S phase with incompletely andor abnormally replicated DNA Tumour cells with a functional intraS phase checkpoint appear to undergo rapid death after replica tion fork stallingcollapse whereas more transformed cancer cells appear to survive longer but eventually face mitotic catastrophe as a result of partially repli cated chromosomes 125126128 In striking contrast normal cells avoid entering S phase with a reduced number of replicationcompetent origins by engaging a recently described cell cycle checkpoint the origin activation checkpoint Several studies have shown that following impairment of the DNA replication initiation machinery normal cells arrest at the G1S boundary with unreplicated DNA elevated p53 levels and induc tion of CDKI p21 111125126 We discovered that the molecular architecture of the underlying cell cycle checkpoint is critically dependent on several tumour suppressor proteins including p53 p21 Dkk3 ARF Hdm2 FoxO3a p15 p27 and RB 129 Figure 4A This suggests that loss of the protective checkpoint mechanism through inactivating mutations in check point proteins will render most common solid tumours sensitive to anticancer agents targeting the DNA repli cation initiation machinery 129 Cdc7 kinase has emerged as a particularly attrac tive anticancer target in the DNA replication initia tion pathway because it can be readily inhibited using ATPcompetitive SMIs Several biopharma companies have initiated Cdc7 drug development programmes some of which have reached earlystage clinical tri als 130131 Firstinclass Cdc7 inhibitors have broad tumour spectrum activity in preclinical models consis tent with loss of the protective checkpoint mechanism in most tumour types Apoptotic cancer cell death in response to Cdc7 inhibition is p53independent and at least in some cancer cell lines is mediated via the stressactivated protein p38MAPK in an ATM and Rad3related ATRdependent manner 132 Interest ingly in addition to its function in origin firing Cdc7 kinase has been shown to play an essential role in mediating the ATRChk1 pathway by phosphorylat ing the Chk1 activator Claspin 133134 Hence the dual effect of Cdc7 inhibitors on DNA replication and DNA damage response pathways may further potenti ate cancer cell killing Importantly expression profile analysis of cell cycle biomarkers in surgical resection specimens provides further target validation for Cdc7 inhibitors and has shown that deregulation of this kinase is linked to aggressive disease For example increased Cdc7 expression in breast cancer is associated with Her2 and triplereceptor negative subtypes the accelerated cell cycle phenotype c arrested tumour differentiation genomic instability increasing NPI score and reduced diseasefree survival 111 Similarly increased Cdc7 expression has been linked with arrested tumour differ entiation advanced clinical stage genomic instability accelerated cell cycle progression and reduced disease free survival in ovarian cancer 118 We postulate that it will be tumours showing the accelerated cell cycle phenotype c high Cdc7 levels and harbouring lesions in the origin activation checkpoint axes that are likely to show optimal response to Cdc7 inhibi tion Thus Cdc7 inhibitors may significantly broaden the therapeutic armamentarium available for the treat ment of aggressive primary and metastatic disease in which treatment options are limited This supposition is Copyright 2011 Pathological Society of Great Britain and Ireland J Pathol 2012 226 352364 Published by John Wiley Sons Ltd wwwpathsocorguk wwwthejournalofpathologycom The cell cycle and cancer 361 supported by preclinical data showing that Cdc7 knock down by RNAi in p53mutant Her2 and triple recep tornegative breast cancer cell lines induces potent cancer cell killing 111 In vivo these breast cancer subtypes are characterized by the accelerated cell cycle phenotype c high Cdc7 expression levels mutation of checkpoint effector proteins and therefore are poten tially sensitive to Cdc7 inhibitors The DNA origin activation checkpoint and cyclotherapy Nucleotide incorporation experiments have demon strated that the G1 arrest triggered by Cdc7 knock down in normal cells is fully reversible on recovery of Cdc7 protein levels and that the arrested cells remain viable 111 This finding suggests that inhibitors tar geting the DNA replication initiation machinery are likely to have limited toxicity in selfrenewing tissues with high turnover eg gut skin or the haemopoietic system compared to conventional cell cyclephase specific agents Indeed in a mouse xenograft Colo 205 tumour model p53mutant cell line we observed potent tumour cell killing after treatment with a Cdc7 SMI while no overt toxic effects were observed in normal mouse gut mucosa Figure 4B Many of the most effective neoadjuvant and adju vant systemic chemotherapeutic regimes utilize S and Mphase agents Table 1 The Achilles heel of these regimes is that S and Mphase agents also affect nor mal cycling cells in particular those located in the tran sit amplifying compartments of selfrenewing tissues resulting in marrow suppression neutropenia hair loss and gut toxicity Following the original cyclotherapy concept as proposed by Arthur Pardee and colleagues treatment with a Cdc7 inhibitor prior to administration of S andor Mphase cell cycle phasespecific agents might provide a powerful method for shielding nor mal somatic cycling cells from the toxic effects of chemotherapy Such combinatorial treatment regimes may allow increased dosage and frequency of cell cycle phasespecific agents thus increasing the therapeutic window and thereby reducing the likelihood of drug resistant clones 135 The availability of Cdc7 SMIs now provides an opportunity for this novel treatment paradigm to be tested Conclusions and implications The cell cycle engine is a promising diagnostic and therapeutic target in cancer because it lies downstream at the convergence point of complex oncogenic sig nalling networks and its deregulation is central to the aberrant cell proliferation that characterizes all cancers Moreover structurally and mechanistically many of its components are evolutionarily conserved and therefore clinical applications are likely to be suited to diverse tumour types This is in stark contrast to the targeting of cancerspecific mutations Many of the fundamen tal discoveries in the cell cycle field have come not from mammalian cells but from genetic and biochem ical studies in yeast Drosophila zebrafish and Xenopus model systems With pathologists traditionally placed at the interface of basic and clinical sciences the study of candidate cell cycle biomarkers in human tissues with linked clinical outcome measures now provides a crucial bridge to translate fundamental discoveries in the cell cycle field into frontline diagnostic and ther apeutic applications Acknowledgment We thank past and present members of the UCL Chromosomal Replication Group and in particular Marco Loddo and Alex Wollenschlaeger for their help with the preparation of the manuscript Author contributions GHW and KS wrote the paper and had final approval of the submitted and published versions References 1 Potten CS Loeffler M Stem cells attributes cycles spirals pit falls and uncertainties Lessons for and from the crypt Develop ment 1990 110 10011020 2 Hall PA Watt FM Stem cells the generation and maintenance of cellular diversity Development 1989 106 619633 3 Williams GH Stoeber K Cell cycle markers in clinical oncology Curr Opin Cell Biol 2007 19 672679 4 Murray A Hunt T The Cell Cycle An Introduction Oxford University Press New York 1993 5 Zetterberg A Larsson O Kinetic analysis of regulatory events in G1 leading to proliferation or quiescence of Swiss 3T3 cells Proc Natl Acad Sci USA 1985 82 53655369 6 Malumbres M Barbacid M Is Cyclin D1CDK4 kinase a bona fide cancer target Cancer Cell 2006 9 24 7 PlanasSilva MD Weinberg RA The restriction point and control of cell proliferation Curr Opin Cell Biol 1997 9 768772 8 Nigg EA Mitotic kinases as regulators of cell division and its checkpoints Nat Rev Mol Cell Biol 2001 2 2132 9 Hartwell LH Weinert TA Checkpoints controls that ensure the order of cell cycle events Science 1989 246 629634 10 Bartek J Lukas C Lukas J Checking on DNA damage in S phase Nat Rev Mol Cell Biol 2004 5 792804 11 Musacchio A Salmon ED The spindleassembly checkpoint in space and time Nat Rev Mol Cell Biol 2007 8 379393 12 Malumbres M Barbacid M Cell cycle CDKs and cancer a changing paradigm Nat Rev Cancer 2009 9 153166 13 Hanahan D Weinberg RA Hallmarks of cancer the next gener ation Cell 2011 144 646674 14 Harbour JW Dean DC The RbE2F pathway expanding roles and emerging paradigms Genes Dev 2000 14 23932409 15 Zhang J Yang PL Gray NS Targeting cancer with small molecule kinase inhibitors Nat Rev Cancer 2009 9 2839 Copyright 2011 Pathological Society of Great Britain and Ireland J Pathol 2012 226 352364 Published by John Wiley Sons Ltd wwwpathsocorguk wwwthejournalofpathologycom 362 GH Williams and K Stoeber 16 Freier K Joos S Flechtenmacher C et al Tissue microarray analysis reveals sitespecific prevalence of oncogene amplifica tions in head and neck squamous cell carcinoma Cancer Res 2003 63 11791182 17 Huang ZY Baldwin RL Hedrick NM et al Astrocytespecific expression of CDK4 is not sufficient for tumor formation but cooperates with p53 heterozygosity to provide a growth advantage for astrocytes in vivo Oncogene 2002 21 13251334 18 Malumbres M Barbacid M To cycle or not to cycle a critical decision in cancer Nat Rev Cancer 2001 1 222231 19 Perou CM Sorlie T Eisen MB et al Molecular portraits of human breast tumours Nature 2000 406 747752 20 vant Veer LJ Dai H van de Vijver MJ et al Gene expression profiling predicts clinical outcome of breast cancer Nature 2002 415 530536 21 vant Veer LJ Bernards R Enabling personalized cancer medicine through analysis of geneexpression patterns Nature 2008 452 564570 22 Michiels S Koscielny S Hill C Prediction of cancer outcome with microarrays a multiple random validation strategy Lancet 2005 365 488492 23 Dunkler D Michiels S Schemper M Gene expression profiling does it add predictive accuracy to clinical characteristics in cancer prognosis Eur J Cancer 2007 43 745751 24 Mackay A Weigelt B Grigoriadis A et al Microarraybased class discovery for molecular classification of breast cancer analysis of interobserver agreement J Natl Cancer Inst 2011 103 662673 25 BuenodeMesquita JM van Harten WH Retel VP et al Use of 70gene signature to predict prognosis of patients with node negative breast cancer a prospective communitybased feasibility study RASTER Lancet Oncol 2007 8 10791087 26 Hannemann J Oosterkamp HM Bosch CA et al Changes in gene expression associated with response to neoadjuvant chemotherapy in breast cancer J Clin Oncol 2005 23 33313342 27 Sotiriou C Piccart MJ Taking geneexpression profiling to the clinic when will molecular signatures become relevant to patient care Nat Rev Cancer 2007 7 545553 28 Weinstein IB Joe AK Mechanisms of disease Oncogene addic tiona rationale for molecular targeting in cancer therapy Nat Clin Pract Oncol 2006 3 448457 29 Stratton MR Campbell PJ Futreal PA The cancer genome Nature 2009 458 719724 30 Sierra JR Cepero V Giordano S Molecular mechanisms of acquired resistance to tyrosine kinase targeted therapy Mol Can cer 2010 9 75 31 Wick W Puduvalli VK Chamberlain MC et al Phase III study of enzastaurin compared with lomustine in the treatment of recurrent intracranial glioblastoma J Clin Oncol 2010 28 11681174 32 Jain RK Duda DG Clark JW et al Lessons from Phase III clinical trials on antiVEGF therapy for cancer Nat Clin Pract Oncol 2006 3 2440 33 Rini BI Halabi S Rosenberg JE et al Phase III trial of beva cizumab plus interferonα versus interferonα monotherapy in patients with metastatic renal cell carcinoma final results of CALGB 90206 J Clin Oncol 2010 28 21372143 34 Fisher WE The promise of a personalized genomic approach to pancreatic cancer and why targeted therapies have missed the mark World J Surg 2011 35 17661769 35 Batchelor T Mulholland P Neyns B et al The efficacy of cedi ranib as monotherapy and in combination with lomustine com pared to lomustine alone in patients with recurrent glioblastoma a Phase III randomized study NeuroOncology 2010 12 iv6978 36 AstraZeneca AstraZeneca provides update on cediranib RECENTIN AZD2171 clinical development programme httpwwwastrazenecauscomsearchitemId2289031 accessed 27 February 2008 37 AstraZeneca AstraZeneca announces results of recentin HORIZON II Phase III trial in metastatic colorectal can cer httpwwwastrazenecacomMediaPressreleasesArticle 20100528AstraZenecaAnnouncesResultsofRecentin HORIZONII accessed 28 May 2010 38 AstraZeneca Phase II data contributes to understanding of cedi ranib in recurrent glioblastoma httpastrazenecausacomabout astrazenecausnewsroomitemId8889831 accessed 14 May 2010 39 Williams GH Romanowski P Morris L et al Improved cer vical smear assessment using antibodies against proteins that regulate DNA replication Proc Natl Acad Sci USA 1998 95 1493214937 40 Williams G Stoeber K Clinical applications of a novel mam malian cellfree DNA replication system Br J Cancer 1999 80suppl 1 2024 41 Gonzalez MA Tachibana KE Laskey RA et al Control of DNA replication and its potential clinical exploitation Nat Rev Cancer 2005 5 135141 42 Blow JJ Gillespie PJ Replication licensing and cancera fatal entanglement Nat Rev Cancer 2008 8 799806 43 Blow JJ Hodgson B Replication licensingdefining the prolif erative state Trends Cell Biol 2002 12 7278 44 Machida YJ Hamlin JL Dutta A Right place right time and only once replication initiation in metazoans Cell 2005 123 1324 45 Masai H Matsumoto S You Z et al Eukaryotic chromosome DNA replication where when and how Annu Rev Biochem 2010 79 89130 46 Sclafani RA Holzen TM Cell cycle regulation of DNA replica tion Annu Rev Genet 2007 41 237280 47 Labib K Diffley JF Is the MCM27 complex the eukaryotic DNA replication fork helicase Curr Opin Genet Dev 2001 11 6470 48 Blow JJ Tanaka TU The chromosome cycle coordinating repli cation and segregation Second in the cycles review series EMBO Rep 2005 6 10281034 49 Perkins G Diffley JF Nucleotidedependent prereplicative com plex assembly by Cdc6p a homolog of eukaryotic and prokaryotic clamploaders Mol Cell 1998 2 2332 50 Labib K How do Cdc7 and cyclindependent kinases trigger the initiation of chromosome replication in eukaryotic cells Genes Dev 2010 24 12081219 51 Sheu YJ Stillman B Cdc7Dbf4 phosphorylates MCM proteins via a docking sitemediated mechanism to promote S phase progression Mol Cell 2006 24 101113 52 Sheu YJ Stillman B The Dbf4Cdc7 kinase promotes S phase by alleviating an inhibitory activity in Mcm4 Nature 2010 463 113117 53 Montagnoli A Valsasina B Brotherton D et al Identification of Mcm2 phosphorylation sites by Sphaseregulating kinases J Biol Chem 2006 281 1028110290 54 Gambus A Jones RC SanchezDiaz A et al GINS maintains association of Cdc45 with MCM in replisome progression com plexes at eukaryotic DNA replication forks Nat Cell Biol 2006 8 358366 55 Labib K Gambus A A key role for the GINS complex at DNA replication forks Trends Cell Biol 2007 17 271278 56 Moyer SE Lewis PW Botchan MR Isolation of the Cdc45Mcm27GINS CMG complex a candidate for Copyright 2011 Pathological Society of Great Britain and Ireland J Pathol 2012 226 352364 Published by John Wiley Sons Ltd wwwpathsocorguk wwwthejournalofpathologycom The cell cycle and cancer 363 the eukaryotic DNA replication fork helicase Proc Natl Acad Sci USA 2006 103 1023610241 57 Blow JJ Dutta A Preventing rereplication of chromosomal DNA Nat Rev Mol Cell Biol 2005 6 476486 58 Li X Zhao Q Liao R et al The SCFSkp2 ubiquitin ligase complex interacts with the human replication licensing factor Cdt1 and regulates Cdt1 degradation J Biol Chem 2003 278 3085430858 59 Tada S Li A Maiorano D et al Repression of origin assembly in metaphase depends on inhibition of RLFBCdt1 by geminin Nat Cell Biol 2001 3 107113 60 Wohlschlegel JA Kutok JL Weng AP et al Expression of gem inin as a marker of cell proliferation in normal tissues and malig nancies Am J Pathol 2002 161 267273 61 Okorokov AL Orlova EV Kingsbury SR et al Molecular struc ture of human geminin Nat Struct Mol Biol 2004 11 10211022 62 Stoeber K Tlsty TD Happerfield L et al DNA replication licensing and human cell proliferation J Cell Sci 2001 114 20272041 63 Eward KL Obermann EC Shreeram S et al DNA replication licensing in somatic and germ cells J Cell Sci 2004 117 58755886 64 Stoeber K Mills AD Kubota Y et al Cdc6 protein causes premature entry into S phase in a mammalian cellfree system EMBO J 1998 17 72197229 65 Mailand N Diffley JF CDKs promote DNA replication origin licensing in human cells by protecting Cdc6 from APCC dependent proteolysis Cell 2005 122 915926 66 Barkley LR Hong HK Kingsbury SR et al Cdc6 is a rate limiting factor for proliferative capacity during HL60 cell dif ferentiation Exp Cell Res 2007 313 37893799 67 Musahl C Holthoff HP Lesch R et al Stability of the replica tive Mcm3 protein in proliferating and differentiating human cells Exp Cell Res 1998 241 260264 68 Freeman A Morris LS Mills AD et al Minichromosome main tenance proteins as biological markers of dysplasia and malig nancy Clin Cancer Res 1999 5 21212132 69 Going JJ Keith WN Neilson L et al Aberrant expression of minichromosome maintenance proteins 2 and 5 and Ki67 in dysplastic squamous oesophageal epithelium and Barretts mucosa Gut 2002 50 373377 70 Alison MR Hunt T Forbes SJ Minichromosome maintenance MCM proteins may be precancer markers Gut 2002 50 290291 71 Ramnath N Hernandez FJ Tan DF et al MCM2 is an indepen dent predictor of survival in patients with nonsmallcell lung cancer J Clin Oncol 2001 19 42594266 72 Meng MV Grossfeld GD Williams GH et al Minichromosome maintenance protein 2 expression in prostate characterization and association with outcome after therapy for cancer Clin Cancer Res 2001 7 27122718 73 Kayes OJ Loddo M Patel N et al DNA replication licensing factors and aneuploidy are linked to tumor cell cycle state and clinical outcome in penile carcinoma Clin Cancer Res 2009 15 73357344 74 Kulkarni AA Loddo M Leo E et al DNA replication licensing factors and aurora kinases are linked to aneuploidy and clinical outcome in epithelial ovarian carcinoma Clin Cancer Res 2007 13 61536161 75 Dudderidge TJ Stoeber K Loddo M et al Mcm2 Geminin and KI67 define proliferative state and are prognostic markers in renal cell carcinoma Clin Cancer Res 2005 11 25102517 76 Korkolopoulou P Givalos N Saetta A et al Minichromosome maintenance proteins 2 and 5 expression in muscleinvasive urothelial cancer a multivariate survival study including prolif eration markers and cell cycle regulators Hum Pathol 2005 36 899907 77 Gonzalez MA Pinder SE Callagy G et al Minichromosome maintenance protein 2 is a strong independent prognostic marker in breast cancer J Clin Oncol 2003 21 43064313 78 Rhodes DR Yu J Shanker K et al Largescale metaanalysis of cancer microarray data identifies common transcriptional profiles of neoplastic transformation and progression Proc Natl Acad Sci USA 2004 101 93099314 79 Yan Z DeGregori J Shohet R et al Cdc6 is regulated by E2F and is essential for DNA replication in mammalian cells Proc Natl Acad Sci USA 1998 95 36033608 80 Yoshida K Inoue I Regulation of Geminin and Cdt1 expression by E2F transcription factors Oncogene 2004 23 38023812 81 Ohtani K Iwanaga R Nakamura M et al Cell growthregulated expression of mammalian MCM5 and MCM6 genes mediated by the transcription factor E2F Oncogene 1999 18 22992309 82 Yoshida K Inoue I Expression of MCM10 and TopBP1 is regulated by cell proliferation and UV irradiation via the E2F transcription factor Oncogene 2004 23 62506260 83 Yamada M Sato N Taniyama C et al A 63base pair DNA segment containing an Sp1 site but not a canonical E2F site can confer growthdependent and E2Fmediated transcriptional stimulation of the human ASK gene encoding the regulatory subunit for human Cdc7related kinase J Biol Chem 2002 277 2766827681 84 Liontos M Koutsami M Sideridou M et al Deregulated over expression of hCdt1 and hCdc6 promotes malignant behavior Cancer Res 2007 67 1089910909 85 Arentson E Faloon P Seo J et al Oncogenic potential of the DNA replication licensing protein CDT1 Oncogene 2002 21 11501158 86 Gonzalez S Klatt P Delgado S et al Oncogenic activity of Cdc6 through repression of the INK4ARF locus Nature 2006 440 702706 87 Seo J Chung YS Sharma GG et al Cdt1 transgenic mice develop lymphoblastic lymphoma in the absence of p53 Onco gene 2005 24 81768186 88 Honeycutt KA Chen Z Koster MI et al Deregulated minichro mosomal maintenance protein MCM7 contributes to oncogene driven tumorigenesis Oncogene 2006 25 40274032 89 Shima N Alcaraz A Liachko I et al A viable allele of Mcm4 causes chromosome instability and mammary adenocarcinomas in mice Nat Genet 2007 39 9398 90 Pruitt SC Bailey KJ Freeland A Reduced Mcm2 expression results in severe stemprogenitor cell deficiency and cancer Stem Cells 2007 25 31213132 91 Kelly D Kincaid E Fansler Z et al Detection of cervical high grade squamous intraepithelial lesions from cytologic samples using a novel immunocytochemical assay ProEx C Cancer 2006 108 494500 92 Shroyer KR Homer P Heinz D et al Validation of a novel immunocytochemical assay for topoisomerase IIα and minichro mosome maintenance protein 2 expression in cervical cytology Cancer 2006 108 324330 93 Depuydt CE Makar AP Ruymbeke MJ et al BDProExC as adjunct molecular marker for improved detection of CIN2 after HPV primary screening Cancer Epidemiol Biomarkers Prev 2011 20 628637 94 Ayaru L Stoeber K Webster GJ et al Diagnosis of pancreatico biliary malignancy by detection of minichromosome maintenance protein 5 in bile aspirates Br J Cancer 2008 98 15481554 95 Dudderidge TJ Kelly JD Wollenschlaeger A et al Diagnosis of prostate cancer by detection of minichromosome maintenance 5 protein in urine sediments Br J Cancer 2010 103 701707 Copyright 2011 Pathological Society of Great Britain and Ireland J Pathol 2012 226 352364 Published by John Wiley Sons Ltd wwwpathsocorguk wwwthejournalofpathologycom 364 GH Williams and K Stoeber 96 Williams GH Swinn R Prevost AT et al Diagnosis of oesophageal cancer by detection of minichromosome maintenance 5 protein in gastric aspirates Br J Cancer 2004 91 714719 97 Stoeber K Halsall I Freeman A et al Immunoassay for urothe lial cancers that detects DNA replication protein Mcm5 in urine Lancet 1999 354 15241525 98 Stoeber K Swinn R Prevost AT et al Diagnosis of genito urinary tract cancer by detection of minichromosome maintenance 5 protein in urine sediments J Natl Cancer Inst 2002 94 10711079 99 Fletcher AH Barklow TA Murphy NJ et al ProExC triage of atypical glandular cells on liquidbased cervical cytology specimens J Low Genit Tract Dis 2011 15 610 100 Tambouret RH Misdraji J Wilbur DC Longitudinal clinical evaluation of a novel antibody cocktail for detection of highgrade squamous intraepithelial lesions on cervical cytology specimens Arch Pathol Lab Med 2008 132 918925 101 Siddiqui MT Cohen C Nassar A Detecting highgrade cervical disease on ASCH cytology role of BD ProEx C and Digene Hybrid Capture II HPV DNA testing Am J Clin Pathol 2008 130 765770 102 Elston CW Ellis IO Pathological prognostic factors in breast cancer I The value of histological grade in breast cancer experi ence from a large study with longterm followup Histopathology 1991 19 403410 103 Coindre JM Grading of soft tissue sarcomas review and update Arch Pathol Lab Med 2006 130 14481453 104 Hugh J Hanson J Cheang MC et al Breast cancer subtypes and response to docetaxel in nodepositive breast cancer use of an immunohistochemical definition in the BCIRG 001 trial J Clin Oncol 2009 27 11681176 105 PenaultLlorca F Andre F Sagan C et al Ki67 expression and docetaxel efficacy in patients with estrogen receptorpositive breast cancer J Clin Oncol 2009 27 28092815 106 Yerushalmi R Woods R Ravdin PM et al Ki67 in breast cancer prognostic and predictive potential Lancet Oncol 2010 11 174183 107 Hook SS Lin JJ Dutta A Mechanisms to control rereplication and implications for cancer Curr Opin Cell Biol 2007 19 663671 108 Crosio C Fimia GM Loury R et al Mitotic phosphorylation of histone H3 spatiotemporal regulation by mammalian Aurora kinases Mol Cell Biol 2002 22 874885 109 Wei Y Yu L Bowen J et al Phosphorylation of histone H3 is required for proper chromosome condensation and segregation Cell 1999 97 99109 110 Loddo M Kingsbury SR Rashid M et al Cellcyclephase pro gression analysis identifies unique phenotypes of major prognostic and predictive significance in breast cancer Br J Cancer 2009 100 959970 111 RodriguezAcebes S Proctor I Loddo M et al Targeting DNA replication before it starts Cdc7 as a therapeutic target in p53 mutant breast cancers Am J Pathol 2010 177 20342045 112 Whitfield ML Sherlock G Saldanha AJ et al Identification of genes periodically expressed in the human cell cycle and their expression in tumors Mol Biol Cell 2002 13 19772000 113 Wirapati P Sotiriou C Kunkel S et al Metaanalysis of gene expression profiles in breast cancer toward a unified understand ing of breast cancer subtyping and prognosis signatures Breast Cancer Res 2008 10 R65 114 Paik S Shak S Tang G et al A multigene assay to predict recurrence of tamoxifentreated nodenegative breast cancer N Engl J Med 2004 351 28172826 115 Proctor I Stoeber K Williams GH Biomarkers in bladder cancer Histopathology 2010 57 113 116 Ellis P BarrettLee P Johnson L et al Sequential docetaxel as adjuvant chemotherapy for early breast cancer TACT an open label phase III randomised controlled trial Lancet 2009 373 16811692 117 Poole CJ Hiller L Howard HC et al tAnGo a randomized Phase III trial of gemcitabine gem in paclitaxelcontaining epirubicincyclophosphamidebased adjuvant chemotherapy CT for women with earlystage breast cancer EBC J Clin Oncol 2008 26 Abstract 506 118 Kulkarni AA Kingsbury SR Tudzarova S et al Cdc7 kinase is a predictor of survival and a novel therapeutic target in epithelial ovarian carcinoma Clin Cancer Res 2009 15 24172425 119 Rinehart J Adjei AA Lorusso PM et al Multicenter Phase II study of the oral MEK inhibitor CI1040 in patients with advanced nonsmallcell lung breast colon and pancreatic can cer J Clin Oncol 2004 22 44564462 120 Wilhelm SM Carter C Tang L et al BAY 439006 exhibits broad spectrum oral antitumor activity and targets the RAFMEKERK pathway and receptor tyrosine kinases involved in tumor progression and angiogenesis Cancer Res 2004 64 70997109 121 Paez JG Janne PA Lee JC et al EGFR mutations in lung cancer correlation with clinical response to gefitinib therapy Science 2004 304 14971500 122 Bild AH Potti A Nevins JR Linking oncogenic pathways with therapeutic opportunities Nat Rev Cancer 2006 6 735741 123 Bild AH Yao G Chang JT et al Oncogenic pathway signatures in human cancers as a guide to targeted therapies Nature 2006 439 353357 124 Huang E Ishida S Pittman J et al Gene expression phenotypic models that predict the activity of oncogenic pathways Nat Genet 2003 34 226230 125 Shreeram S Sparks A Lane DP et al Cell typespecific responses of human cells to inhibition of replication licensing Oncogene 2002 21 66246632 126 Montagnoli A Tenca P Sola F et al Cdc7 inhibition reveals a p53dependent replication checkpoint that is defective in cancer cells Cancer Res 2004 64 71107116 127 Montagnoli A Valsasina B Croci V et al A Cdc7 kinase inhibitor restricts initiation of DNA replication and has antitumor activity Nat Chem Biol 2008 4 357365 128 Ge XQ Jackson DA Blow JJ Dormant origins licensed by excess Mcm27 are required for human cells to survive replica tive stress Genes Dev 2007 21 33313341 129 Tudzarova S Trotter MW Wollenschlaeger A et al Molecular architecture of the DNA replication origin activation checkpoint EMBO J 2010 29 33813394 130 Swords R Mahalingam D ODwyer M et al Cdc7 kinasea new target for drug development Eur J Cancer 2010 46 3340 131 Montagnoli A Moll J Colotta F Targeting cell division cycle 7 kinase a new approach for cancer therapy Clin Cancer Res 2010 16 45034508 132 Im JS Lee JK ATRdependent activation of p38 MAP kinase is responsible for apoptotic cell death in cells depleted of Cdc7 J Biol Chem 2008 283 2517125177 133 Kim JM Kakusho N Yamada M et al Cdc7 kinase mediates Claspin phosphorylation in DNA replication checkpoint Onco gene 2008 27 34753482 134 Matsumoto S Shimmoto M Kakusho N et al Hsk1 kinase and Cdc45 regulate replication stressinduced checkpoint responses in fission yeast Cell Cycle 2010 9 46274637 135 Blagosklonny MV Pardee AB Exploiting cancer cell cycling for selective protection of normal cells Cancer Res 2001 61 43014305 Copyright 2011 Pathological Society of Great Britain and Ireland J Pathol 2012 226 352364 Published by John Wiley Sons Ltd wwwpathsocorguk wwwthejournalofpathologycom