David A. Gillespie

The ATM–Chk2 and ATR–Chk1 Pathways in DNA Damage Signaling and Cancer

DNA damage is a key factor both in the evolution and treatment of cancer. Genomic instability is a common feature of cancer cells, fuelling accumulation of oncogenic mutations, while radiation and diverse genotoxic agents remain important, if imperfect, therapeutic modalities. Cellular responses to DNA damage are coordinated primarily by two distinct kinase signaling cascades, the ATM-Chk2 and ATR-Chk1 pathways, which are activated by DNA double-strand breaks (DSBs) and single-stranded DNA respectively. Historically, these pathways were thought to act in parallel with overlapping functions; however, more recently it has become apparent that their relationship is more complex. In response to DSBs, ATM is required both for ATR-Chk1 activation and to initiate DNA repair via homologous recombination (HRR) by promoting formation of single-stranded DNA at sites of damage through nucleolytic resection. Interestingly, cells and organisms survive with mutations in ATM or other components required for HRR, such as BRCA1 and BRCA2, but at the cost of genomic instability and cancer predisposition. By contrast, the ATR-Chk1 pathway is the principal direct effector of the DNA damage and replication checkpoints and, as such, is essential for the survival of many, although not all, cell types. Remarkably, deficiency for HRR in BRCA1- and BRCA2-deficient tumors confers sensitivity to cisplatin and inhibitors of poly(ADP-ribose) polymerase (PARP), an enzyme required for repair of endogenous DNA damage. In addition, suppressing DNA damage and replication checkpoint responses by inhibiting Chk1 can enhance tumor cell killing by diverse genotoxic agents. Here, we review current understanding of the organization and functions of the ATM-Chk2 and ATR-Chk1 pathways and the prospects for targeting DNA damage signaling processes for therapeutic purposes.

Mutant p53 Drives Invasion by Promoting Integrin Recycling

p53 is a tumor suppressor protein whose function is frequently lost in cancers through missense mutations within the Tp53 gene. This results in the expression of point-mutated p53 proteins that have both lost wild-type tumor suppressor activity and show gain of functions that contribute to transformation and metastasis. Here, we show that mutant p53 expression can promote invasion, loss of directionality of migration, and metastatic behavior. These activities of p53 reflect enhanced integrin and epidermal growth factor receptor (EGFR) trafficking, which depends on Rab-coupling protein (RCP) and results in constitutive activation of EGFR/integrin signaling. We provide evidence that mutant p53 promotes cell invasion via the inhibition of TAp63, and simultaneous loss of p53 and TAp63 recapitulates the phenotype of mutant p53 in cells. These findings open the possibility that blocking alpha5/beta1-integrin and/or the EGF receptor will have therapeutic benefit in mutant p53-expressing cancers.

Chk1-deficient tumour cells are viable but exhibit multiple checkpoint and survival defects

The conserved protein kinase Chk1 is believed to play an important role in checkpoint responses to aberrant DNA structures; however, genetic analysis of Chk1 functions in metazoans is complicated by lethality of Chk1‐deficient embryonic cells. We have used gene targeting to eliminate Chk1 function in somatic DT40 B‐lymphoma cells. We find that Chk1‐deficient DT40 cells are viable, but fail to arrest in G2/M in response to and are hypersensitive to killing by ionizing radiation. Chk1‐deficient cells also fail to maintain viable replication forks or suppress futile origin firing when DNA polymerase is inhibited, leading to incomplete genome duplication and diminished cell survival after release from replication arrest. In contrast to embryonic cells, however, Chk1 is not required to delay mitosis when DNA synthesis is inhibited. Thus, Chk1 is dispensable for normal cell division in somatic DT40 cells but is essential for DNA damage‐induced G2/M arrest and a subset of replication checkpoint responses. Furthermore, Chk1‐dependent processes promote tumour cell survival after perturbations of DNA structure or metabolism.

Chk1 regulates the density of active replication origins during the vertebrate S phase

The checkpoint kinase 1 (Chk1) preserves genome integrity when replication is performed on damaged templates. Recently, Chk1 has also been implicated in regulating different aspects of unperturbed S phase. Using mammalian and avian cells with compromised Chk1 activity, we show that an increase in active replicons compensates for inefficient DNA polymerisation. In the absence of damage, loss of Chk1 activity correlates with the frequent stalling and, possibly, collapse of active forks and activation of adjacent, previously suppressed, origins. In human cells, super‐activation of replication origins is restricted to pre‐existing replication factories. In avian cells, in contrast, Chk1 deletion also correlates with the super‐activation of replication factories and loss of temporal continuity in the replication programme. The same phenotype is induced in wild‐type avian cells when Chk1 or ATM/ATR is inhibited. These observations show that Chk1 regulates replication origin activation and contributes to S‐phase progression in somatic vertebrate cells.

Molecular mechanism and biological functions of c-Jun N-terminal kinase signalling via the c-Jun transcription factor

The regulation of c-Jun transcriptional activity by Jun N-terminal kinase (JNK) has become a paradigm for understanding how mitogen-activated protein (MAP) kinase signalling pathways elicit specific changes in gene transcription through selective phosphorylation of nuclear transcription factors. Selective phosphorylation of c-Jun by JNK is determined by a specific docking motif in c-Jun, the delta region, which enables JNK to associate physically with c-Jun. Analogous MAP kinase docking motifs have subsequently been found in several other transcription factors, indicating that this is a general mechanism for ensuring specificity of signal transduction. Genetic and biochemical studies in mice, flies and cultured cells have provided evidence that signals relayed by JNK through c-Jun regulate a range of cellular processes including cell proliferation, tumourigenesis, apoptosis and embryonic development. Despite these advances, in most cases, the genes or programs of gene expression downstream of JNK and c-Jun, which control these processes, have not been defined. Here, we review the current understanding of the molecular basis and biological consequences of JNK signalling via c-Jun and highlight some of the mechanistic issues, which remain to be resolved.

Chk1 Requirement for High Global Rates of Replication Fork Progression during Normal Vertebrate S Phase

ABSTRACT Chk1 protein kinase maintains replication fork stability in metazoan cells in response to DNA damage and DNA replication inhibitors. Here, we have employed DNA fiber labeling to quantify, for the first time, the extent to which Chk1 maintains global replication fork rates during normal vertebrate S phase. We report that replication fork rates in Chk1 −/− chicken DT40 cells are on average half of those observed with wild-type cells. Similar results were observed if Chk1 was inhibited or depleted in wild-type DT40 cells or HeLa cells by incubation with Chk1 inhibitor or small interfering RNA. In addition, reduced rates of fork extension were observed with permeabilized Chk1 −/− cells in vitro. The requirement for Chk1 for high fork rates during normal S phase was not to suppress promiscuous homologous recombination at replication forks, because inhibition of Chk1 similarly slowed fork progression in XRCC3 −/− DT40 cells. Rather, we observed an increased number of replication fibers in Chk1 −/− cells in which the nascent strand is single-stranded, supporting the idea that slow global fork rates in unperturbed Chk1 −/− cells are associated with the accumulation of aberrant replication fork structures.

DNA damage induces Chk1-dependent centrosome amplification

Centrosomal abnormalities are frequently observed in cancers and in cells with defective DNA repair. Here, we used light and electron microscopy to show that DNA damage induces centrosome amplification, not fragmentation, in human cells. Caffeine abrogated this amplification in both ATM (ataxia telangiectasia, mutated)‐ and ATR (ATM and Rad3‐related)‐defective cells, indicating a complementary role for these DNA‐damage‐responsive kinases in promoting centrosome amplification. Inhibition of checkpoint kinase 1 (Chk1) by RNA‐mediated interference or drug treatment suppressed DNA‐damage‐induced centrosome amplification. Radiation‐induced centrosome amplification was abrogated in Chk1−/− DT40 cells, but occurred at normal levels in Chk1−/− cells transgenically expressing Chk1. Expression of kinase‐dead Chk1, or Chk1S345A, through which the phosphatidylinositol‐3‐kinase cannot signal, failed to restore centrosome amplification, showing that signalling to Chk1 and Chk1 catalytic activity are necessary to promote centrosome overduplication after DNA damage.

High levels of phosphorylated c-Jun, Fra-1, Fra-2 and ATF-2 proteins correlate with malignant phenotypes in the multistage mouse skin carcinogenesis model.

Analysis of the functions of AP-1 transcription factor in cellular systems has shown its key role as a mediator of oncogenic signals. The employment of suitable animal model systems greatly facilitates the study of changes in the composition and activity of the AP-1 complex. Here, we have analysed the quantitative and qualitative changes of AP-1 at different stages of carcinogenesis in mouse skin cell lines, derived from tumours induced by chemical mutagens. The findings of this study suggest that elevated AP-1 DNA binding and transactivation activity characterize the carcinoma cell lines, most notably the highly malignant spindle carcinomas. In addition, increased amounts and post-translational modifications of c-Jun, Fra-1, Fra-2 and ATF-2 proteins account for a high percentage of the increased AP-1 activity. Remarkably, high levels of phosphorylated ATF-2 protein were detected in malignant cell lines, indicating a novel role of ATF-2 in tumour progression. c-Jun and ATF-2 proteins are phosphorylated by highly active JNK kinases present in tumour cells. Finally, our results indicate distinct functions for different AP-1 components in the promotion and progression of mouse skin tumours.

A new c-Jun N-terminal kinase (JNK)-interacting protein, Sab (SH3BP5), associates with mitochondria.

We have identified a novel c-Jun N-terminal kinase (JNK)-interacting protein, Sab, by yeast two-hybrid screening. Sab binds to and serves as a substrate for JNK in vitro, and was previously found to interact with the Src homology 3 (SH3) domain of Brutons tyrosine kinase (Btk). Inspection of the sequence of Sab reveals the presence of two putative mitogen-activated protein kinase interaction motifs (KIMs) similar to that found in the JNK docking domain of the c-Jun transcription factor, and four potential serine-proline JNK phosphorylation sites in the C-terminal half of the molecule. Using deletion and site-directed mutagenesis, we demonstrate that the most N-terminal KIM in Sab is essential for JNK binding, and that, as with c-Jun, physical interaction with JNK is necessary for Sab phosphorylation. Interestingly, confocal immunocytochemistry and cell fractionation studies indicate that Sab is associated with mitochondria, where it co-localizes with a fraction of active JNK. These and previously reported properties of Sab suggest a possible role in targeting JNK to this subcellular compartment and/or mediating cross-talk between the Btk and JNK signal transduction pathways.

Chk1-Dependent S-M Checkpoint Delay in Vertebrate Cells Is Linked to Maintenance of Viable Replication Structures

ABSTRACT We investigated mitotic delay during replication arrest (the S-M checkpoint) in DT40 B-lymphoma cells deficient in the Chk1 or Chk2 kinase. We show here that cells lacking Chk1, but not those lacking Chk2, enter mitosis with incompletely replicated DNA when DNA synthesis is blocked, but only after an initial delay. This initial delay persists when S-M checkpoint failure is induced in Chk2−/− cells with the Chk1 inhibitor UCN-01, indicating that it does not depend on Chk1 or Chk2 activity. Surprisingly, dephosphorylation of tyrosine 15 did not accompany Cdc2 activation during premature entry to mitosis in Chk1−/− cells, although mitotic phosphorylation of cyclin B2 did occur. Previous studies have shown that Chk1 is required to stabilize stalled replication forks during replication arrest, and strikingly, premature mitosis occurs only in Chk1-deficient cells which have lost the capacity to synthesize DNA as a result of progressive replication fork inactivation. These results suggest that Chk1 maintains the S-M checkpoint indirectly by preserving the viability of replication structures and that it is the continued presence of such structures, rather than the activation of Chk1 per se, which delays mitosis until DNA replication is complete.