DNA Damage-induced G2 Checkpoint Research Articles

G2 checkpoint targeting via Wee1 inhibition radiosensitizes EGFRvIII-positive glioblastoma cells

BackgroundThe gene of the Epidermal growth factor receptor (EGFR) is one of the most frequently altered genes in glioblastoma (GBM), with deletions of exons 2–7 (EGFRvIII) being amongst the most common genomic mutations. EGFRvIII is heterogeneously expressed in GBM. We already showed that EGFRvIII expression has an impact on chemosensitivity, replication stress, and the DNA damage response. Wee1 kinase is a major regulator of the DNA damage induced G2 checkpoint. It is highly expressed in GBM and its overexpression is associated with poor prognosis. Since Wee1 inhibition can lead to radiosensitization of EGFRvIII-negative (EGFRvIII−) GBM cells, we asked, if Wee1 inhibition is sufficient to radiosensitize also EGFRvIII-positive (EGFRvIII+) GBM cells.MethodsWe used the clinically relevant Wee1 inhibitor adavosertib and two pairs of isogenetic GBM cell lines with and without endogenous EGFRvIII expression exhibiting different TP53 status. Moreover, human GBM samples displaying heterogenous EGFRvIII expression were analyzed. Expression of Wee1 was assessed by Western blot and respectively immunohistochemistry. The impact of Wee1 inhibition in combination with irradiation on cell cycle and cell survival was analyzed by flow cytometry and colony formation assay.ResultsAnalysis of GBM cells and patient samples revealed a higher expression of Wee1 in EGFRvIII+ cells compared to their EGFRvIII− counterparts. Downregulation of EGFRvIII expression by siRNA resulted in a strong decrease in Wee1 expression. Wee1 inhibition efficiently abrogated radiation-induced G2-arrest and caused radiosensitization, without obvious differences between EGFRvIII− and EGFRvIII+ GBM cells.ConclusionWe conclude that the inhibition of Wee1 is an effective targeting approach for the radiosensitization of both EGFRvIII− and EGFRvIII+ GBM cells and may therefore represent a promising new therapeutic option to increase response to radiotherapy.

P53 suppresses SENP3 phosphorylation to mediate G2 checkpoint

In response to DNA damage, p53-mediated signaling is regulated by protein phosphorylation and ubiquitination to precisely control G2 checkpoint. Here we demonstrated that protein SUMOylation also engaged in regulation of p53-mediated G2 checkpoint. We found that G2 DNA damage suppressed SENP3 phosphorylation at G2/M phases in p53-dependent manner. We further found that the suppression of SENP3 phosphorylation was crucial for efficient DNA damage/p53-induced G2 checkpoint and G2 arrest. Mechanistically, we identified Cdh1, a subunit of APC/C complex, was a SUMOylated protein at G2/M phase. SENP3 could de-SUMOylate Cdh1. DNA damage/p53-induced suppression of SENP3 phosphorylation activated SENP3 de-SUMOylation of Cdh. De-SUMOylation promoted Cdh1 de-phosphorylation by phosphatase Cdc14B, and then activated APC/CCdh1 E3 ligase activity to ubiquitate and degrade Polo-like kinase 1 (Plk1) in process of G2 checkpoint. These data reveal that p53-mediated inhibition of SENP3 phosphorylation regulates the activation of Cdc14b-APC/CCdh1-Plk1 axis to control DNA damage-induced G2 checkpoint.

Acetylation of MORC2 by NAT10 regulates cell-cycle checkpoint control and resistance to DNA-damaging chemotherapy and radiotherapy in breast cancer.

MORC family CW-type zinc finger 2 (MORC2) is an oncogenic chromatin-remodeling enzyme with an emerging role in DNA repair. Here, we report a novel function for MORC2 in cell-cycle checkpoint control through an acetylation-dependent mechanism. MORC2 is acetylated by the acetyltransferase NAT10 at lysine 767 (K767Ac) and this process is counteracted by the deacetylase SIRT2 under unperturbed conditions. DNA-damaging chemotherapeutic agents and ionizing radiation stimulate MORC2 K767Ac through enhancing the interaction between MORC2 and NAT10. Notably, acetylated MORC2 binds to histone H3 phosphorylation at threonine 11 (H3T11P) and is essential for DNA damage-induced reduction of H3T11P and transcriptional repression of its downstream target genes CDK1 and Cyclin B1, thus contributing to DNA damage-induced G2 checkpoint activation. Chemical inhibition or depletion of NAT10 or expression of an acetylation-defective MORC2 (K767R) forces cells to pass through G2 checkpoint, resulting in hypersensitivity to DNA-damaging agents. Moreover, MORC2 acetylation levels are associated with elevated NAT10 expression in clinical breast tumor samples. Together, these findings uncover a previously unrecognized role for MORC2 in regulating DNA damage-induced G2 checkpoint through NAT10-mediated acetylation and provide a potential therapeutic strategy to sensitize breast cancer cells to DNA-damaging chemotherapy and radiotherapy by targeting NAT10.

Hypoxia-induced alterations of G2 checkpoint regulators

Hypoxia promotes an aggressive tumor phenotype with increased genomic instability, partially due to downregulation of DNA repair pathways. However, genome stability is also surveilled by cell cycle checkpoints. An important issue is therefore whether hypoxia also can influence the DNA damage-induced cell cycle checkpoints. Here, we show that hypoxia (24 h 0.2% O2) alters the expression of several G2 checkpoint regulators, as examined by microarray gene expression analysis and immunoblotting of U2OS cells. While some of the changes reflected hypoxia-induced inhibition of cell cycle progression, the levels of several G2 checkpoint regulators, in particular Cyclin B, were reduced in G2 phase cells after hypoxic exposure, as shown by flow cytometric barcoding analysis of individual cells. These effects were accompanied by decreased phosphorylation of a Cyclin dependent kinase (CDK) target in G2 phase cells after hypoxia, suggesting decreased CDK activity. Furthermore, cells pre-exposed to hypoxia showed increased G2 checkpoint arrest upon treatment with ionizing radiation. Similar results were found following other hypoxic conditions (∼0.03% O2 20 h and 0.2% O2 72 h). These results demonstrate that the DNA damage-induced G2 checkpoint can be altered as a consequence of hypoxia, and we propose that such alterations may influence the genome stability of hypoxic tumors.

Phospho-Bcl-xL(Ser62) plays a key role at DNA damage-induced G2 checkpoint

Accumulating evidence suggests that Bcl-xL, an anti-apoptotic member of the Bcl-2 family, also functions in cell cycle progression and cell cycle checkpoints. Analysis of a series of phosphorylation site mutants reveals that cells expressing Bcl-xL(Ser62Ala) mutant are less stable at the G2 checkpoint and enter mitosis more rapidly than cells expressing wild-type Bcl-xL or Bcl-xL phosphorylation site mutants, including Thr41Ala, Ser43Ala, Thr47Ala, Ser56Ala and Thr115Ala. Analysis of the dynamic phosphorylation and location of phospho-Bcl-xL(Ser62) in unperturbed, synchronized cells and during DNA damage-induced G2 arrest discloses that a pool of phospho-Bcl-xL(Ser62) accumulates into nucleolar structures in etoposide-exposed cells during G2 arrest. In a series of in vitro kinase assays, pharmacological inhibitors and specific siRNAs experiments, we found that Polo kinase 1 and MAPK9/JNK2 are major protein kinases involved in Bcl-xL(Ser62) phosphorylation and accumulation into nucleolar structures during the G2 checkpoint. In nucleoli, phospho-Bcl-xL(Ser62) binds to and co-localizes with Cdk1(cdc2), the key cyclin-dependent kinase required for entry into mitosis. These data indicate that during G2 checkpoint, phospho-Bcl-xL(Ser62) stabilizes G2 arrest by timely trapping of Cdk1(cdc2) in nucleolar structures to slow mitotic entry. It also highlights that DNA damage affects the dynamic composition of the nucleolus, which now emerges as a piece of the DNA damage response.

B-MYB Is Required for Recovery from the DNA Damage–Induced G2 Checkpoint in p53 Mutant Cells

In response to DNA damage, several signaling pathways that arrest the cell cycle in G(1) and G(2) are activated. The down-regulation of mitotic genes contributes to the stable maintenance of the G(2) arrest. The human LINC or DREAM complex, together with the B-MYB transcription factor, plays an essential role in the expression of G(2)-M genes. Here, we show that DNA damage results in the p53-dependent binding of p130 and E2F4 to LINC and the dissociation of B-MYB from LINC. We find that B-MYB fails to dissociate from LINC in p53 mutant cells, that this contributes to increased G(2)-M gene expression in response to DNA damage in these cells, and, importantly, that B-MYB is required for recovery from the G(2) DNA damage checkpoint in p53-negative cells. Reanalysis of microarray expression data sets revealed that high levels of B-MYB correlate with a p53 mutant status and an advanced tumor stage in primary human breast cancer. Taken together, these data suggest that B-MYB/LINC plays an important role in the DNA damage response downstream of p53.

Germ cell specific protein VASA is over-expressed in epithelial ovarian cancer and disrupts DNA damage-induced G2 checkpoint

ObjectiveCancer cells have characteristics, such as high telomerase activity and high levels of migration activity and proliferation, which are very similar to those of germ cell lineages. In this study, we examined the expression of VASA, a germ cell lineage specific marker and evaluated its clinical significance in epithelial ovarian cancer (EOC). MethodsWe investigated VASA expression in 75 EOC tissues by immunohistochemistry, correlating results with clinicopathological factors. To clarify the effects of VASA on cellular phenotypes, we compared the protein expression profiles between SKOV-3 cells stably expressing VASA (SKOV-3-VASA) and vector-control cell lines by coupling 2D fingerprinting and identification of proteins by mass spectrometry. ResultsVASA expression in tumor cells was found in 21of 75 cases and was positively correlated with high age and serous histology. Significant down-regulation of 14-3-3σ was observed in SKOV-3-VASA versus control cells. Over-expression of VASA abrogates the G2 checkpoint, induced by DNA damage, by down-regulating the expression of 14-3-3σ. ConclusionsThese results suggest that VASA may either play a direct role in the progression of EOC or serve as a valuable marker of tumorigenesis.

P53-Dependent Chk1 Phosphorylation is Required for Maintenance of Prolonged G2Arrest

Targeting checkpoint kinases has been shown to have a potential chemosensitizing effect in cancer treatment. However, inhibitors of such kinases preferentially abrogate the DNA damage-induced G2 checkpoint in p53-/- as opposed to p53+/+ cells. The mechanisms by which p53 (TP53) can prevent abrogation of the G2 checkpoint are unclear. Using normal human diploid p53+/+ and p53-/- fibroblasts as model systems, we have compared the effects of three checkpoint inhibitors, caffeine, staurosporine and UCN-01, on gamma-radiation-induced G2 arrest. The G2 arrest in p53+/+ cells was abrogated by caffeine, but not by staurosporine and UCN-01, whereas the G2 arrest in p53-/- cells was sensitive to all three inhibitors. Chk2 (CHEK1) phosphorylation was maintained in the presence of all three inhibitors in both p53+/+ and p53-/- cells. Chk1 phosphorylation was maintained only in the presence of staurosporine and UCN-01 in p53+/+ cells. In the presence of caffeine Chk1 phosphorylation was inhibited regardless of p53 status. The pathway of Chk1 phosphorylation --> Cdc25A degradation --> inhibition of cyclin B1/Cdk1 activity --> G2 arrest is accordingly resistant to staurosporine and UCN-01 in p53+/+ cells. Moreover, sustained phosphorylation of Chk1 in the presence of staurosporine and UCN-01 is strongly related to phosphorylation of p53. The present study suggests the unique role of Chk1 in preventing abrogation of the G2 checkpoint in p53+/+ cells.

A Stronger DNA Damage-Induced G2 Checkpoint Due to Over-Activated CHK1 in the Absence of PARP-1

Poly(ADP-ribose) polymerase-1 (PARP-1) is involved in multi-pathways to respond to DNA damage. Lack of or inhibition of PARP-1 activity leads to slow progress of cell cycle and sensitization of cells to different stresses. Recently, it was reported that besides the Ku- dependent main non-homologous end joining (NHEJ) pathway, there is a PARP-1-dependent complementary NHEJ pathway to repair DNA double strand break (DSB). Here we show that compared with PARP-1+/+ cells, PARP-1-/- cells display a much stronger G2 checkpoint response following ionizing radiation (IR). Treatment with Chk1 siRNA abolishes the stronger G2 checkpoint response and sensitizes PARP-1-/- cells to IR. These data indicate that the stronger G2 checkpoint response in PARP-1-/- cells is CHK1-dependent, which protects cells from IR-induced killing. We also show that 4-Amino-1,8-naphthalimide (4-AN, inhibitor of PARP) but not methoxyamine (inhibitor of base excision repair (BER)), affects IR-induced G2 arrest and cell sensitivity in PARP-1+/+ cells, resulting in the phenotypes similar to those of PARP-1-/- cells. These results indicate that DSB repair from the complementary NHEJ pathway of PARP-1, but not single strand break (SSB) repair from the BER function of PARP-1, may play an essential role in the over-activated CHK1 regulated G2 checkpoint response and radiosensitivity in PARP-1-/- cells.

A Role for the Fanconi Anemia C Protein in Maintaining the DNA Damage-induced G2 Checkpoint

Fanconi anemia (FA) is a complex, heterogeneous genetic disorder composed of at least 11 complementation groups. The FA proteins have recently been found to functionally interact with the cell cycle regulatory proteins ATM and BRCA1; however, the function of the FA proteins in cell cycle control remains incompletely understood. Here we show that the Fanconi anemia complementation group C protein (Fancc) is necessary for proper function of the DNA damage-induced G2/M checkpoint in vitro and in vivo. Despite apparently normal induction of the G2/M checkpoint after ionizing radiation, murine and human cells lacking functional FANCC did not maintain the G2 checkpoint as compared with wild-type cells. The increased rate of mitotic entry seen in Fancc-/-mouse embryo fibroblasts correlated with decreased inhibitory phosphorylation of cdc2 kinase on tyrosine 15. An increased inability to maintain the DNA damage-induced G2 checkpoint was observed in Fancc -/-; Trp53 -/-cells compared with Fancc -/-cells, indicating that Fancc and p53 cooperated to maintain the G2 checkpoint. In contrast, genetic disruption of both Fancc and Atm did not cooperate in the G2 checkpoint. These data indicate that Fancc and p53 in separate pathways converge to regulate the G2 checkpoint. Finally, fibroblasts lacking FANCD2 were found to have a G2 checkpoint phenotype similar to FANCC-deficient cells, indicating that FANCD2, which is activated by the FA complex, was also required to maintain the G2 checkpoint. Because a proper checkpoint function is critical for the maintenance of genomic stability and is intricately related to the function and integrity of the DNA repair process, these data have implications in understanding both the function of FA proteins and the mechanism of genomic instability in FA.

Functional analysis of APC-Cdh1.

To maintain genomic integrity against various kinds of genotoxic stress, cells have multiple checkpoints in the cell cycle. When one of the cell cycle events, such as DNA synthesis, DNA repair, and chromosomal segregation, has not been successfully completed, checkpoints will delay progression until the step is correctly accomplished, and only then will they relieve the arrest to allow the cell to move to the next phase. Cells lacking functional checkpoints display genomic aberrations, resulting in the acquisition of phenotypic changes of cancer cells. Anaphase-promoting complex (APC) is activated by two regulatory proteins: Cdc20 and Cdh1. In yeast and Drosophila, Cdh1-dependent APC (APC-Cdh1) activity targets mitotic cyclins from the end of mitosis to the G1 phase. Loss of Cdh1 induces unscheduled accumulation of mitotic cyclins in G1, resulting in abrogation of G1 arrest caused by treatment with rapamycin, an inducer of p27Kip1. Furthermore, Cdh1-deficient DT40 cells fail to maintain DNA damage-induced G2 arrest, and Cdh1-APC is activated by X-irradiation-induced DNA damage. Thus, activation of Cdh1-APC plays a crucial role in both cdk inhibitor-dependent G1 arrest and DNA damage-induced G2 arrest. In light of the differences between normal and cancer cells, checkpoints can be ideal targets for developmental cancer therapeutics. In this chapter, we describe how to analyze three checkpoints (spindle assembly checkpoint, rapamycin-induced G1 checkpoint, and DNA damage-induced G2 checkpoint) in conjunction with cell synchronization.

Roles of aurora-A kinase in mitotic entry and G2 checkpoint in mammalian cells.

Various mitotic events are controlled by Cdc2-cyclin B and other mitotic kinases. Aurora/Ipl1-related mitotic kinases were proved to play key roles in mitotic progression in diverse lower organisms. Aurora-A is a mammalian counterpart of aurora/Ipl1-related kinases and is thought to be a potential oncogene. However, the regulation of aurora-A activation and the commitment of aurora-A in the progression of G2-M phase are largely unknown in mammalian cells. We demonstrated that aurora-A is activated depending on the activation of Cdc2-cyclin B in mammalian cells. Since Cdc2-cyclin B does not directly phosphorylate aurora-A, indirect pathways such as the inhibition of PP1 by Cdc2-cyclin B may act for the activation of aurora-A kinase. Microinjection of anti-aurora-A antibodies into HeLa cells at late G2 phase caused a significant delay in mitotic entry. Furthermore, aurora-A activation at G2-M transition was inhibited by DNA damage, and the over-expression of aurora-A induced the abrogation of the DNA damage-induced G2 checkpoint. Aurora-A is activated downstream of Cdc2-cyclin B and plays crucial roles in proper mitotic entry and G2 checkpoint control. Dysregulation of aurora-A induces abnormal G2-M transition in mammalian cells and may lead to chromosome instability, which results in the development and progression of malignant tumours.

Activation of Cdh1-dependent APC is required for G1 cell cycle arrest and DNA damage-induced G2 checkpoint in vertebrate cells

Anaphase-promoting complex (APC) is activated by two regulatory proteins, Cdc20 and Cdh1. In yeast and Drosophila, Cdh1-dependent APC (Cdh1-APC) activity targets mitotic cyclins from the end of mitosis to the G1 phase. To investigate the function of Cdh1 in vertebrate cells, we generated clones of chicken DT40 cells disrupted in their Cdh1 loci. Cdh1 was dispensable for viability and cell cycle progression. However, similarly to yeast and Drosophila, loss of Cdh1 induced unscheduled accumulation of mitotic cyclins in G1, resulting in abrogation of G1 arrest caused by treatment with rapamycin, an inducer of p27(Kip1). Further more, we found that Cdh1(-/-) cells fail to maintain DNA damage-induced G2 arrest and that Cdh1-APC is activated by X-irradiation-induced DNA damage. Thus, activation of Cdh1-APC plays a crucial role in both cdk inhibitor-dependent G1 arrest and DNA damage-induced G2 arrest.

Lack of effect of caffeine post-treatment on X-ray-induced chromosomal aberrations in Werner's syndrome lymphoblastoid cell lines: a preliminary report

Purpose: To investigate whether in Werner's syndrome cells the G2 phase of the cell cycle has some abnormal response to post-treatment with agents such as caffeine and hydroxyurea known to interfere with cellular response to DNA damage. Materials and methods: Two Werner's syndrome lymphoblastoid cell lines (KO375 and DJG) and the normal cell line SNW646 were exposed to 50cGy of X-rays or mitomycin-C and post-treated with caffeine or hydroxyurea in the G2 phase of the cell cycle. Results: Hydroxyurea post-treatment potentiated the X-ray-induced aberration levels both in the normal and Werner's syndrome (KO375 and DJG) cell lines; in contrast caffeine was only effective in the normal cell line. Similar results were observed when Werner's syndrome cells were treated in the G1 phase with the S-dependent agent mitomycin-C and post-treated with caffeine in G2, extending the observation that Werner's syndrome cells are unaffected by caffeine G2 post-treatment. Conclusions: These results show a lack of caffeine effect in Werner's syndrome cells, suggesting an involvement of the Werner's syndrome protein in the signal transduction pathway by which caffeine could override the DNA damage induced G2 checkpoint.

Nuclear export of cyclin B1 and its possible role in the DNA damage-induced G2 checkpoint.

M-phase-promoting factor (MPF), a complex of cdc2 and a B-type cyclin, is a key regulator of the G2/M cell cycle transition. Cyclin B1 accumulates in the cytoplasm through S and G2 phases and translocates to the nucleus during prophase. We show here that cytoplasmic localization of cyclin B1 during interphase is directed by its nuclear export signal (NES)-dependent transport mechanism. Treatment of HeLa cells with leptomycin B (LMB), a specific inhibitor of the NES-dependent transport, resulted in nuclear accumulation of cyclin B1 in G2 phase. Disruption of an NES which has been identified in cyclin B1 here abolished the nuclear export of this protein, and consequently the NES-disrupted cyclin B1 when expressed in cells accumulated in the nucleus. Moreover, we show that expression of the NES-disrupted cyclin B1 or LMB treatment of the cells is able to override the DNA damage-induced G2 checkpoint when combined with caffeine treatment. These results suggest a role of nuclear exclusion of cyclin B1 in the DNA damage-induced G2 checkpoint.

Evidence for DNA-PK-dependent and -independent DNA double-strand break repair pathways in mammalian cells as a function of the cell cycle.

Mice homozygous for the scid (severe combined immune deficiency) mutation are defective in the repair of DNA double-strand breaks (DSBs) and are consequently very X-ray sensitive and defective in the lymphoid V(D)J recombination process. Recently, a strong candidate for the scid gene has been identified as the catalytic subunit of the DNA-dependent protein kinase (DNA-PK) complex. Here, we show that the activity of the DNA-PK complex is regulated in a cell cycle-dependent manner, with peaks of activity found at the G1/early S phase and again at the G2 phase in wild-type cells. Interestingly, only the deficit of the G1/early S phase DNA-PK activity correlated with an increased hypersensitivity to X-irradiation and a DNA DSB repair deficit in synchronized scid pre-B cells. Finally, we demonstrate that the DNA-PK activity found at the G2 phase may be required for exit from a DNA damage-induced G2 checkpoint arrest. These observations suggest the presence of two pathways (DNA-PK-dependent and -independent) of illegitimate mammalian DNA DSB repair and two distinct roles (DNA DSB repair and G2 checkpoint traversal) for DNA-PK in the cellular response to ionizing radiation.