DNA replication licensing and human cell proliferation

Kai Stoeber 0 1 2 Thea D. Tlsty 4 Lisa Happerfield 2 Geraldine A. Thomas 3 Sergei Romanov 4 Lynda Bobrow 2 E. Dillwyn Williams 3 Gareth H. Williams 2 0 Department of Zoology, University of Cambridge , Downing Street, Cambridge CB2 3EJ , UK 1 Wellcome/CRC Institute, University of Cambridge , Tennis Court Road, Cambridge CB2 1QR , UK 2 Department of Pathology, University of Cambridge , Tennis Court Road, Cambridge CB2 1QP , UK 3 Thyroid Carcinogenesis Group, University of Cambridge, Strangeways Research Laboratory , UK 4 Department of Pathology, University of California San Francisco, School of Medicine , CA 94143-0506 , USA SUMMARY The convergence point of growth regulatory pathways that control cell proliferation is the initiation of genome replication, the core of which is the assembly of prereplicative complexes resulting in chromatin being licensed for DNA replication in the subsequent S phase. We have analysed regulation of the pre-replicative complex proteins ORC, Cdc6, and MCM in cycling and nonproliferating quiescent, differentiated and replicative senescent human cells. Moreover, a human cell-free DNA replication system has been exploited to study the replicative capacity of nuclei and cytosolic extracts prepared from these cells. These studies demonstrate that downregulation of the Cdc6 and MCM constituents of the replication initiation pathway is a common downstream mechanism for loss of proliferative capacity in human cells. Furthermore, analysis of MCM protein expression in selfReplication of mammalian genomes is an integrated step of the cell division cycle that is strictly regulated by an intricate network of extracellular and intracellular signalling pathways that control cell proliferation, quiescence, differentiation, cellular senescence and apoptosis. The convergence point of these complex growth regulatory pathways is the initiation of genome replication, the core of which is a set of replication initiation factors that sequentially assemble into pre-replicative complexes (pre-RCs) at replication origins resulting in chromatin being licensed for replication in the subsequent S phase (reviewed by Ritzi and Knippers, 2000). The current understanding of how replication initiation is controlled in eukaryotic cells is derived mainly from studies in yeast and Xenopus (reviewed by Donaldson and Blow, 1999; Tye, 1999). However, the constituents of the pre-RC have been shown to be conserved from yeast to mammals, suggesting that the basic mechanisms of replication initiation are very similar in all eukaryotic cells (reviewed by Dutta and Bell, 1997). The consensus view that has emerged is that pre-RC assembly begins with the binding of a six-subunit origin recognition complex (ORC) to specific origin sites in the genome (reviewed by Quintana and Dutta, 1999). ORC determines where replication initiation will occur and serves as a landing platform for additional initiation factors during a cascade of renewing, stable and permanent human tissues shows that the three classes of tissue have developed very different growth control strategies with respect to replication licensing. Notably, in breast tissue we found striking differences between the proportion of mammary acinar cells that express MCM proteins and those labelled with conventional proliferation markers, raising the intriguing possibility that progenitor cells of some tissues are held in a prolonged G1 phase or in-cycle arrest. We conclude that biomarkers for replication-licensed cells detect, in addition to actively proliferating cells, cells with growth potential, a concept that has major implications for developmental and cancer biology. protein assembly that finally results in the formation of the preRC (Fig. 1). In early G1, the Cdc6 protein functionally interacts with ORC and loads the MCM proteins (Mcm2-7), which were originally discovered and named as factors that support minichromosome maintenance in yeast (reviewed by Tye, 1999), onto chromatin. The assembly of ORC, Cdc6 and MCM proteins into pre-RCs makes chromatin competent or licensed for replication (reviewed by Chevalier and Blow, 1996). Cdc6 is released just before or at the beginning of S phase and replaced by the Cdc45 protein, which, as a first step in establishing replication forks, recruits DNA polymerase a -primase and the single-strand-specific DNA-binding protein RPA to the replication origin (reviewed by Ritzi and Knippers, 2000). At the G1 to S phase transition, DNA replication is initiated by the concerted action of S phase-promoting cyclin-dependent kinases and the Dbf4-dependent Cdc7 kinase (reviewed by Pasero and Schwob, 2000; Sclafani, 2000). The MCM proteins gradually dissociate from chromatin as S phase proceeds (Krude et al., 1996), consistent with their predicted function as a DNA helicase (Ishimi, 1997). Dissociation of Cdc6 and MCM proteins from chromatin ensures that DNA is replicated once and only once during a single cell division cycle, as replicated chromatin is devoid of functional pre-RCs and thus not licensed for replication (reviewed by Stillman, 1996). Although recent reports confirm the universality of this scheme, they also reveal that the regulation of cell division in multicellular organisms imposes an additional level of complexity. In higher eukaryotes, cells divide not only to reproduce themselves, but also to ensure the correct formation of tissues and organs, requiring differential regulation of growth. In mammalian tissues the majority of cells have exited the cell division cycle during growth and development into different out-of-cycle states. First, normal somatic cells require the presence of mitogens for continual proliferation. After the removal of mitogens in early/mid G1 prior to progression through the restriction point untransformed cells cease proliferation and reversibly withdraw into a quiescent state (G0) in which macromolecular synthesis is reduced (Pardee, 1989). Second, proliferating precursor and progenitor cells can be induced by both intrinsic and extrinsic signals to stop dividing and to enter the differentiation pathway (reviewed by Hall and Watt, 1989). Often there is mutual antagonism between the cellular circuits that control proliferation and differentiation (reviewed by Myster and Duronio, 2000). Third, normal somatic cells have a finite replicative lifespan that restricts cell division by a process known as replicative or cellular senescence. Normal cells proliferate for a finite number of cell divisions after which they withdraw irreversibly from the cell cycle, entering a senescent state (Hayflick and Moorhead, 1961). Senescent cells remain viable indefinitely but fail to initiate DNA replication in response to mitogens (reviewed by Campisi, 1996). The molecular mechanisms that underlie loss of proliferative capacity as cells withdraw from the cell division cycle are largely unknown. Moreover, the ability to arrest growth in quiescence, terminal differentiation and (...truncated)