Cisplatin, carboplatin and oxaliplatin are platinum-based drugs that are widely used in cancer chemotherapy. Platinum–DNA adducts, which are formed following uptake of the drug into the nucleus of cells, activate several cellular processes that mediate the cytotoxicity of these platinum drugs. This review focuses on recently discovered cellular pathways that are activated in response to cisplatin, including those involved in regulating drug uptake, the signalling of DNA damage, cell-cycle checkpoints and arrest, DNA repair and cell death. Such knowledge of the cellular processing of cisplatin adducts with DNA provides valuable clues for the rational design of more efficient platinum-based drugs as well as the development of new therapeutic strategies.

Cellular processing of platinum anticancer drugs.

The inability to repair DNA damage properly in mammals leads to various disorders and enhanced rates of tumour development. Organisms respond to chromosomal insults by activating a complex damage response pathway. This pathway regulates known responses such as cell-cycle arrest and apoptosis (programmed cell death), and has recently been shown to control additional processes including direct activation of DNA repair networks.

https://faculty.missouri.edu/~gatesk/DNADamageResponse.pdf

The DNA damage response: putting checkpoints in perspective

DNA damage is a relatively common event in the life of a cell and may lead to mutation, cancer, and cellular or organismic death. Damage to DNA induces several cellular responses that enable the cell either to eliminate or cope with the damage or to activate a programmed cell death process, presumably to eliminate cells with potentially catastrophic mutations. These DNA damage response reactions include: (a) removal of DNA damage and restoration of the continuity of the DNA duplex; (b) activation of a DNA damage checkpoint, which arrests cell cycle progression so as to allow for repair and prevention of the transmission of damaged or incompletely replicated chromosomes; (c) transcriptional response, which causes changes in the transcription profile that may be beneficial to the cell; and (d) apoptosis, which eliminates heavily damaged or seriously deregulated cells. DNA repair mechanisms include direct repair, base excision repair, nucleotide excision repair, double-strand break repair, and cross-link repair. The DNA damage checkpoints employ damage sensor proteins, such as ATM, ATR, the Rad17-RFC complex, and the 9-1-1 complex, to detect DNA damage and to initiate signal transduction cascades that employ Chk1 and Chk2 Ser/Thr kinases and Cdc25 phosphatases. The signal transducers activate p53 and inactivate cyclin-dependent kinases to inhibit cell cycle progression from G1 to S (the G1/S checkpoint), DNA replication (the intra-S checkpoint), or G2 to mitosis (the G2/M checkpoint). In this review the molecular mechanisms of DNA repair and the DNA damage checkpoints in mammalian cells are analyzed.

Molecular Mechanisms of Mammalian DNA Repair and the DNA Damage Checkpoints

To ensure the high-fidelity transmission of genetic information, cells have evolved mechanisms to monitor genome integrity. Cells respond to DNA damage by activating a complex DNA-damage-response pathway that includes cell-cycle arrest, the transcriptional and post-transcriptional activation of a subset of genes including those associated with DNA repair, and, under some circumstances, the triggering of programmed cell death. An inability to respond properly to, or to repair, DNA damage leads to genetic instability, which in turn may enhance the rate of cancer development. Indeed, it is becoming increasingly clear that deficiencies in DNA-damage signaling and repair pathways are fundamental to the etiology of most, if not all, human cancers. Here we describe recent progress in our understanding of how cells detect and signal the presence and repair of one particularly important form of DNA damage induced by ionizing radiation-the DNA double-strand break (DSB). Moreover, we discuss how tumor suppressor proteins such as p53, ATM, Brca1 and Brca2 have been linked to such pathways, and how accumulating evidence is connecting deficiencies in cellular responses to DNA DSBs with tumorigenesis.

DNA double-strand breaks: signaling, repair and the cancer connection.

The genomes of eukaryotic cells are under continuous assault by environmental agents (e.g., UV light and reactive chemicals) as well as the byproducts of normal intracellular metabolism (e.g., reactive oxygen intermediates and inaccurately replicated DNA). Whatever the origin, genetic damage threatens cell survival, and, in metazoans, leads to organ failure, immunodeficiency, cancer, and other pathologic sequelae. To ensure that cells pass accurate copies of their genomes on to the next generation, evolution has overlaid the core cell-cycle machinery with a series of surveillance pathways termed cell-cycle checkpoints. The overall function of these checkpoints is to detect damaged or abnormally structured DNA, and to coordinate cell-cycle progression with DNA repair. Typically, cell-cycle checkpoint activation slows or arrests cell-cycle progression, thereby allowing time for appropriate repair mechanisms to correct genetic lesions before they are passed on to the next generation of daughter cells. In certain cell types, such as thymocytes, checkpoint proteins link DNA strand breaks to apoptotic cell death via induction of p53. Hence, loss of either of two biochemically connected checkpoint kinases, ATM or Chk2, paradoxically increases the resistance of immature (CD4CD8) T cells to ionizing radiation (IR)-induced apoptosis (Xu and Baltimore 1996; Hirao et al. 2000). In a broader context, cell-cycle checkpoints can be envisioned as signal transduction pathways that link the pace of key cell-cycle phase transitions to the timely and accurate completion of prior, contingent events. It is important to recognize that checkpoint surveillance functions are not confined solely to the happenings within the nucleus–extranuclear parameters, such as growth factor availability and cell mass accumulation, also govern the pace of the cell cycle (Stocker and Hafen 2000). However, for the purposes of this review we will focus exclusively on the subset of checkpoints that monitor the status and structure of chromosomal DNA during cell-cycle progression (Fig. 1). These checkpoints contain, as their most proximal signaling elements, sensor proteins that scan chromatin for partially replicated DNA, DNA strand breaks, or other abnormalities, and translate these DNA-derived stimuli into biochemical signals that modulate the functions of specific downstream target proteins. Despite the recent explosion of information regarding the molecular components of cell-cycle checkpoints in eukaryotic cells, we still have only a skeletal understanding of both the identities of the DNA damage sensors and the mechanisms through which they initiate and terminate the activation of checkpoints. However, members of the Rad group of checkpoint proteins, which include Rad17, Rad1, Rad9, Rad26, and Hus1 (nomenclature based on the Schizosaccharomyces pombe gene products) are widely expressed in all eukaryotic cells, and are prime suspects in the lineup of candidate DNA damage sensors (Green et al. 2000; O’Connell et al. 2000). Three of these Rad proteins, Rad1, Rad9, and Hus1, exhibit structural similarity to the proliferating cell nuclear antigen (PCNA), and accumulating evidence supports the idea that this similarity may extend to function as well (Thelen et al. 1999; Burtelow et al. 2000). During DNA replication, PCNA forms a homotrimeric complex that encircles DNA, creating a “sliding clamp” that tethers DNA polymerase to the DNA strand. Rad1, Rad9, and Hus1 are also found as a heterotrimeric complex in intact cells, and it has been postulated that the Rad1–Rad9–Hus1 complex encircles DNA at or near sites of damage to form a checkpoint sliding clamp (CSC) (O’Connell et al. 2000), which could serve as a nucleus for the recruitment of the checkpoint signaling machinery to broken or abnormally structured DNA. The analogy between PCNA and the Rad1–Rad9–Hus1 complex extends even further. The loading of the PCNA clamp onto DNA is controlled by the clamp loading complex, replication factor C (RFC). Interestingly, yet another member of the Rad family, Rad17, bears homology to the RFC subunits and, in fact, associates with RFC subunits to form a putative checkpoint clamp loading complex (CLC) that governs the interaction of the Rad1– Rad9–Hus1 CSC with damaged DNA (Green et al. 2000; O’Connell et al. 2000). Although this model is fascinating, rigorous biochemical evaluations of the interplay between the CLC and CSC complexes, and the interactions of both complexes with damaged chromatin, are needed before the model can be accepted without reservation. Present address: The Burnham Institute, 10901 N. Torrey Pines Road, La Jolla, CA 92037, USA E-MAIL abraham@burnham.org; FAX (858) 713-6268. Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/ gad.914401.

/pdf/cell-cycle-checkpoint-signaling-through-the-atm-and-atr-1vf7u1uv6o.pdf

Cell cycle checkpoint signaling through the ATM and ATR kinases

In response to DNA damage, mammalian cells prevent cell cycle progression through the control of critical cell cycle regulators. A human gene was identified that encodes the protein Chk1, a homolog of the Schizosaccharomyces pombe Chk1 protein kinase, which is required for the DNA damage checkpoint. Human Chk1 protein was modified in response to DNA damage. In vitro Chk1 bound to and phosphorylated the dual-specificity protein phosphatases Cdc25A, Cdc25B, and Cdc25C, which control cell cycle transitions by dephosphorylating cyclin-dependent kinases. Chk1 phosphorylates Cdc25C on serine-216. As shown in an accompanying paper by Peng et al. in this issue, serine-216 phosphorylation creates a binding site for 14-3-3 protein and inhibits function of the phosphatase. These results suggest a model whereby in response to DNA damage, Chk1 phosphorylates and inhibits Cdc25C, thus preventing activation of the Cdc2-cyclin B complex and mitotic entry.

http://science.sciencemag.org/content/sci/277/5331/1497.full.pdf

Conservation of the Chk1 checkpoint pathway in mammals: Linkage of DNA damage to Cdk regulation through Cdc25

The gene coding for human cyclin K was isolated as a CPR (cell-cycle progression restoration) gene by virtue of its ability to impart a Far− phenotype to the budding yeast Saccharomyces cerevisiae and to rescue the lethality of a deletion of the G1 cyclin genes CLN1, CLN2, and CLN3. The cyclin K gene encodes a 357-amino-acid protein most closely related to human cyclins C and H, which have been proposed to play a role in regulating basal transcription through their association with and activation of cyclin-dependent kinases (Cdks) that phosphorylate the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase II (RNAP II). Murine and Drosophila melanogaster homologs of cyclin K have also been identified. Cyclin K mRNA is ubiquitously expressed in adult mouse and human tissues, but is most abundant in the developing germ cells of the adult testis and ovaries. Cyclin K is associated with potent CTD kinase and Cdk kinase (CAK) activity in vitro and coimmunoprecipitates with the large subunit of RNAP II. Thus, cyclin K represents a new member of the “transcription” cyclin family which may play a dual role in regulating Cdk and RNAP II activity.

Human Cyclin K, a Novel RNA Polymerase II-Associated Cyclin Possessing Both Carboxy-Terminal Domain Kinase and Cdk-Activating Kinase Activity

Characterization of a human and murine gene (CLCN3) sharing similarities to voltage-gated chloride channels and to a yeast integral membrane protein.

Kallmann syndrome is an inherited disorder characterized by an abnormality in olfactory system development. The gene for the X-linked form of this disorder (KAL) maps to Xp22.3 and encodes a protein sharing homologies with molecules involved in neuronal migration and axonal pathfinding. Here we report the expression pattern of the KAL gene in various parts of the human fetal brain. We found KAL transcripts in granule cells of the olfactory bulb and the cerebellum, in the dorsomedial thalamus and in the developing cerebral cortex. To determine whether or not signals from the olfactory nerve are required for KAL expression in the olfactory bulb, we analyzed chick embryos in which the olfactory placode was surgically removed. Those embryos lacking an olfactory nerve had a histologically abnormal bulb which nevertheless expressed the KAL gene at high levels. These findings indicate that, while the development of the proper cytoarchitecture of the olfactory bulb requires the innervation by olfactory axons, the expression of KAL is independent of such developmental processes.

Calvin Wong

Papers

Conservation of the Chk1 checkpoint pathway in mammals: Linkage of DNA damage to Cdk regulation through Cdc25

Human Cyclin K, a Novel RNA Polymerase II-Associated Cyclin Possessing Both Carboxy-Terminal Domain Kinase and Cdk-Activating Kinase Activity

Characterization of a human and murine gene (CLCN3) sharing similarities to voltage-gated chloride channels and to a yeast integral membrane protein.

Expression of the Kallmann syndrome gene in human fetal brain and in the manipulated chick embryo