Chk2 is required for optimal mitotic delay in response to irradiation-induced DNA damage incurred in G2 phase.

Whether Chk2 contributes to DNA damage-induced arrest in G2 has been controversial. To investigate this issue further, we generated Chk2-deficient DT40 B-lymphoma cells by gene targeting and compared their cell cycle response to ionizing radiation (IR) with wild-type (WT) and isogenic Chk1-deficient counterparts. After moderate doses of IR (4 Gy), we find that Chk2-/- cells which are in G1 or S phase at the time of irradiation arrest efficiently in G2. In contrast, Chk2-/- cells which are in G2 when DNA damage is incurred exhibit an impaired mitotic delay compared to WT, with the result that cells enter mitosis with damaged DNA as judged by the presence of numerous gamma-H2AX foci on condensed chromosomes. Impaired G2 delay as the result of Chk2 deficiency can be detected at very low doses of radiation (0.1 Gy), and may allow division with spontaneous DNA damage, since a higher proportion of mitotic Chk2-/- cells bear spontaneous gamma-H2AX foci and damaged chromosomes during unperturbed growth compared to WT. The contribution of Chk2 to G2/M delay is epistatic to that of Chk1, since Chk1-/- cells exhibit no measurable mitotic delay at any radiation dose tested. We suggest that this function of Chk2 could contribute to tumour suppression, since cell division with low levels of spontaneous damage is likely to promote genetic instability and thus carcinogenesis.

Chk1-dependent slowing of S-phase progression protects DT40 B-lymphoma cells against killing by the nucleoside analogue 5-fluorouracil.

Chk1 plays a crucial role in the DNA damage and replication checkpoints in vertebrates and may therefore be an important determinant of tumour cell responses to genotoxic anticancer drugs. To evaluate this concept we compared the effects of the nucleoside analogue 5-fluorouracil (5FU) on cell cycle progression and clonogenic survival in DT40 B-lymphoma cells with an isogenic mutant derivative in which Chk1 function was ablated by gene targeting. We show that 5FU activates Chk1 in wild-type DT40 cells and that 5FU-treated cells accumulate in the S phase of the cell cycle due to slowing of the overall rate of DNA replication. In marked contrast, Chk1-deficient DT40 cells fail to slow DNA replication upon initial exposure to 5FU, despite equivalent inhibition of the target enzyme thymidylate synthase, and instead accumulate progressively in the G1 phase of the following cell cycle. This G1 accumulation cannot be reversed rapidly by exogenous thymidine or removal of 5FU, and is associated with increased incorporation of 5FU into genomic DNA and severely diminished clonogenic survival. Taken together, these results demonstrate that a Chk1-dependent replication checkpoint which slows S phase progression can protect tumour cells against the cytotoxic effects of 5FU.

Viral mutations enhance the Max binding properties of the vMyc b-HLH-LZ domain.

Interaction with Max via the helix-loop-helix/leucine zipper (HLH-LZ) domain is essential for Myc to function as a transcription factor. Myc is commonly upregulated in tumours, however, its activity can also be potentiated by virally derived mutations. vMyc, derived from the virus, MC29 gag-Myc, differs from its cellular counterpart by five amino acids. The N-terminal mutation stabilizes the protein, however, the significance of the other mutations is not known. We now show that vMyc can sustain longer deletions in the LZ domain than cMyc before complete loss in transforming activity, implicating the viral mutations in contributing to Myc:Max complex formation. We confirmed this both in vitro and in vivo, with loss of Max binding correlating with a loss in the biological activity of Myc. A specific viral mutation, isoleucine383>leucine (I383>L) in helix 2 of the HLH domain, extends the LZ domain from four to five heptad repeats. Significantly, introduction of I383>L into a Myc mutant that is defective for Max binding substantially restored its ability to complex with Max in vitro and in vivo. We therefore propose that this virally derived mutation is functional by significantly contributing to establishing a more hydrophobic interface between the LZs of Myc and Max.

Sab (SH3BP5), a novel mitochondria-localized JNK-interacting protein.

The JNK (c-Jun N-terminal kinase) pathway is activated by diverse stresses and can have an effect on a number of different cellular processes. Protein-protein interactions are critical for efficient signalling from JNK to multiple targets; through a screen for interacting proteins, we identified a novel JNK-interacting protein, Sab (SH3BP5). Sab has previously been found to interact with the Src homology 3 domain of Bruton's tyrosine kinase; however, the interaction with JNK occurs through a mitogen-activated protein KIM (kinase interaction motif) in a region distinct from the Bruton's tyrosine kinase-binding domain. As with c-Jun, the presence of this KIM is essential for Sab to act as a JNK substrate. Interestingly, Sab is associated with the mitochondria and co-localizes with a portion of active JNK after stress treatment. The present study and previously reported work may suggest a possible role for Sab in targeting JNK to this subcellular compartment and/or mediating crosstalk between different signal-transduction pathways.

v-Jun stimulates both cdk2 kinase activity and G1/S progression via transcriptional repression of p21 CIP1.

Previous studies have shown that the viral Jun (v-Jun) oncoprotein induces marked alterations in cell cycle control, which are associated with, and may be caused by, increased cdk2 kinase activity. Since p21 CIP1 is an important regulator of cdk2, we investigated whether aberrant expression of this cyclin-dependent kinase inhibitor might contribute to cell cycle deregulation by v-Jun. We find that the basal levels of p21 CIP1 mRNA and protein expression are greatly reduced in chick embryo fibroblasts (CEF) transformed by v-Jun, and that v-Jun blocks the increases in p21 CIP1 expression that normally accompany growth inhibition induced by serum deprivation or confluency in untransformed CEF. Importantly, ectopic expression of p21 CIP1 in v-Jun-transformed CEF inhibits both cdk2 kinase activity and cell cycle progression, indicating that these alterations in p21 CIP1 expression are likely to be functionally significant for growth deregulation. We also investigated the mechanism through which v-Jun disturbs p21 CIP1 expression and the possible involvement of a known p21 CIP1 regulator, p53, as an intermediate in this process. This analysis revealed that repression is mediated primarily at the level of p21 CIP1 gene transcription, however the mechanism is complex; both p53-dependent and -independent mechanisms contribute as judged by analysis of p21 CIP1 promoter mutants and other assays of p53 transcriptional activity.