Oncogene (2003) 22, 5834–5847 & 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00 www.nature.com/onc DNA damage checkpoint control in cells exposed to ionizing radiation George Iliakis*,1, Ya Wang2, Jun Guan2 and Huichen Wang2 1Institute of Medical Radiation Biology, University of Essen Medical School, Hufelanstrasse 55, 45122 Essen, Germany; 2Department of Radiation Oncology, Kimmel Cancer Center, Jefferson Medical College, Philadelphia, PA 19107, USA Damage induced in the DNA after exposure of cells to Lu¨ cke-Huhle, 1982). Strong support for this alternative ionizing radiation activates checkpoint pathways that came with the observation that cells from ataxia inhibit progression of cells through the G1 and G2 phases telagiectasia (AT) patients have significantly reduced and induce a transient delay in the progression through delays in their progression through the cell cycle and are, S phase.Checkpoints together with repair and apoptosis at the same time, sensitive to killing after exposure to IR are integrated in a circuitry that determines the ultimate (Houldsworth and Lavin, 1980; Painter and Young, response of a cell to DNA damage.Checkpoint activation 1980; Painter, 1981, 1986; Lavin and Schroeder, 1988). typically requires sensors and mediators of DNA damage, Decisive in the further development of the field was signal transducers and effectors.Here, we review the the isolation, first in the budding and later in the fission current state of knowledge regarding mechanisms of yeast, of repair-proficient, radiation-sensitive mutants checkpoint activation and proteins involved in the different showing defects in radiation-induced cell cycle delays steps of the process.Emphasis is placed on the role of (Lee and Nurse, 1988; Hartwell and Weinert, 1989; ATM and ATR, as well on CHK1 and CHK2 kinases in Nurse, 1990; Norbury and Nurse, 1992; Hartwell and checkpoint response.The roles of downstream effectors, Kastan, 1994; Paulovich et al., 1997). Extensive genetic such as P53 and the CDC25 family of proteins, are also studies with these organisms uncovered a complex described, and connections between repair and checkpoint network of genes that cooperate to delay the normal activation are attempted.The role of checkpoints in progression through the cycle as soon as damage is genomic stability and the potential of improving the registered in the genome. We now know that in these treatment of cancer by DNA damage inducing agents genome-surveillance mechanisms, delay in cell cycle through checkpoint abrogation are also briefly outlined. progression is but one manifestation, captured by the Oncogene (2003) 22, 5834–5847. doi:10.1038/sj.onc.1206682 term DNA damage checkpoint. The present state of knowledge allows the integration of delays induced after Keywords: checkpoints; DNA repair; DNA double- irradiation in the different phases of the cell cycle under strand breaks; ionizing radiation the rubric of a single checkpoint activated by damage in the DNA (Elledge, 1996; Zhou and Elledge, 2000). A parallel development that proved essential for the molecular understanding of the DNA damage check- point was the delineation of events underlying progres- Introduction sion of cells through the cycle. It is now established that the cell cycle is controlled by interdependent regulatory It has been known for over 50 years that exposure of transitions that bring the DNA to a state competent for cells to ionizing radiation (IR) delays the normal duplication and division (Nurse, 1994; Elledge, 1996; progression through the cell cycle (Maity et al., 1994; Sherr, 1996; Stillman, 1996). These transitions are Bernhard et al., 1995; Iliakis, 1997). While a delay in G2 achieved by the activation of cyclin-dependent kinases is the most evident, significant delays also occur in G1, (CDKs), the operation of proteolytic pathways and as well as throughout S phase. These cell cycle delays alterations in the state of chromatin. CDKs are essential were initially interpreted as passive cellular responses components of the cell cycle machinery and are subject resulting from the induction by IR of damage in the to strict regulation by independent mechanisms includ- DNA. However, these early studies also provided ing association with cognate cyclins, phosphorylation at circumstantial evidence that the delays actually reflect specific serine/threonine or tyrosine residues and the induction of cellular processes assisting the irradiated association with specific inhibitory proteins. Not sur- cell to cope with the induced damage by somehow prisingly, CDKs turn out to also function as key targets facilitating repair (Walters et al., 1974; Tobey, 1975; of the checkpoint response. Progression through the cycle mediated by the above cell cycle machinery is ‘checked’ by surveillance mechanisms to ensure that cells will not progress to the next phase of the cell cycle *Correspondence: Professor Dr G Iliakis; before events of the preceding phase have been E-mail: [email protected] completed (Hartwell and Weinert, 1989; Hartwell and DNA damage checkpoint control in cells G Iliakis et al 5835 Kastan, 1994; Nurse, 1997; Paulovich et al., 1997). Conceptual Organization of These surveillance mechanisms, known as cell cycle DNA Damage Checkpoint Response checkpoints, are conceptually distinct from the DNA damage checkpoint. However, as our understanding of Signal the molecular mechanisms underlying the different DNA Damage checkpoints increases important similarities become (Before or after processing) apparent. Here, we briefly outline the current state of knowledge of the DNA damage checkpoint. The progress made since the initial radiobiological observations is specta- Sensors cular and represents the combined achievements of genetics and biochemistry in yeast, Xenopus and REPAIR Signal mammalian cell systems. The review mainly focuses on Transduction the DNA damage checkpoint as presently delineated in Transducers higher eukaryotes exposed to IR. Studies with other systems are only mentioned when the results have direct relevance to higher eukaryotes. Despite the recent dramatic accumulation of knowledge, key issues of Effectors checkpoint response remain unresolved and are dis- cussed briefly. Apoptosis Like many fields in modern biology, the field of checkpoints is nurtured by the conviction that detailed Interphase to Cell Cycle, or Transcription knowledge of the molecular mechanisms will enable a better understanding of the processes underlying geno- Figure 1 Conceptual organization of the essential components mic instability and cancer. Virtually every gene im- and the outcomes of checkpoint responses plicated in the DNA damage checkpoint contributes to genomic stability or is associated with genetic disorders Table 1 or predisposition to cancer development. Moreover, because checkpoint abrogation seems to be an impor- Function Class Gene tant feature of the cancer cell, considerable effort is Sensors/mediators RFC1-like RAD17 presently placed towards the development of com- RFC2-5 pounds that specifically abrogate the remaining check- PCNA-like RAD9 point responses in cancer cells. It is hoped that these RAD1 reagents will significantly improve our ability to combat HUS1 BRCT-containing BRCA1 cancer, alone or in combination with other treatment 53BP1 modalities such as IR. TopBP1 MDC1 DSB recognition/repair Mre11 General concepts and key players RAD50 NBS1 The DNA damage checkpoint can be defined as a network of interacting pathways operating in concert to Transducers PI3 kinases like protein ATR recognize damage in the DNA and elicit the response ATM (Elledge, 1996; Zhou and Elledge, 2000; Nyberg et al., PIKK binding proteins ATRIP Effector kinases CHK1 2002). It shares characteristics of a signal transduction CHK2 pathway, and the participating proteins can be formally divided into sensors, transducers and effectors (Figure 1). Sensor proteins recognize DNA damages, detailed mechanisms of signal transmission and arrest in directly or indirectly, and function to signal the presence the different phases of the cell cycle are described in the of these abnormalities and initiate the biochemical subsequent sections. cascade. Transducers are typically protein kinases that It comes as a surprise that from the basic steps of the relay and amplify the damage signal from the sensors by DNA damage checkpoint, the one involved in the initial phosphorylating other kinases or downstream target sensing of abnormal DNA structures is the less well proteins. Effector proteins include the ultimate down- understood. Two proteins were initially considered good stream targets of the transducer protein kinases. candidates as sensors owing to their ability to be directly Modification of effector proteins by upstream kinases, activated by single- and double-strand breaks (DSBs), directly or indirectly, mediates the inhibition in cell cycle respectively, poly(ADP-ribose) polymerase (PARP) and progression. The effector stage is where the DNA DNA-dependent protein kinase (DNA-PK) (Lu¨ cke- damage checkpoint, interphases with the cell cycle Huhle, 1982; Anderson and Carter, 1996; Lees-Miller, machinery. Proteins presently implicated as sensors 1996; Jeggo, 1997; D’Amours et al., 1999; Smith and and signal transducers are summarized in Table 1, and Jackson, 1999; Smith, 2001; Ziegler and Oei, 2001). key players are described next. Effector proteins and However, they do not seem to be required for the Oncogene DNA damage checkpoint control in cells G Iliakis et al 5836 initiation