Shift in G1-Checkpoint from ATM-Alone to a Cooperative ATM Plus ATR Regulation with Increasing Dose of Radiation

The current view of the involvement of PI3-kinases in checkpoint responses after DNA damage is that ATM is the key regulator of G1-, S- or G2-phase checkpoints, that ATR is only partly involved in the regulation of S- and G2-phase checkpoints and that DNA-PKcs is not involved in checkpoint regulation. However, further analysis of the contributions of these kinases to checkpoint responses in cells exposed to ionizing radiation (IR) recently uncovered striking integrations and interplays among ATM, ATR and DNA-PKcs that adapt not only to the phase of the cell cycle in which cells are irradiated, but also to the load of DNA double-strand breaks (DSBs), presumably to optimize their processing. Specifically, we found that low IR doses in G2-phase cells activate a G2-checkpoint that is regulated by epistatically coupled ATM and ATR. Thus, inhibition of either kinase suppresses almost fully its activation. At high IR doses, the epistatic ATM/ATR coupling relaxes, yielding to a cooperative regulation. Thus, single-kinase inhibition suppresses partly, and only combined inhibition suppresses fully G2-checkpoint activation. Interestingly, DNA-PKcs integrates with ATM/ATR in G2-checkpoint control, but functions in its recovery in a dose-independent manner. Strikingly, irradiation during S-phase activates, independently of dose, an exclusively ATR-dependent G2 checkpoint. Here, ATM couples with DNA-PKcs to regulate checkpoint recovery. In the present work, we extend these studies and investigate organization and functions of these PI3-kinases in the activation of the G1 checkpoint in cells irradiated either in the G0 or G1 phase. We report that ATM is the sole regulator of the G1 checkpoint after exposure to low IR doses. At high IR doses, ATM remains dominant, but contributions from ATR also become detectable and are associated with limited ATM/ATR-dependent end resection at DSBs. Under these conditions, only combined ATM + ATR inhibition fully abrogates checkpoint and resection. Contributions of DNA-PKcs and CHK2 to the regulation of the G1 checkpoint are not obvious in these experiments and may be masked by the endpoint employed for checkpoint analysis and perturbations in normal progression through the cell cycle of cells exposed to DNA-PKcs inhibitors. The results broaden our understanding of organization throughout the cell cycle and adaptation with increasing IR dose of the ATM/ATR/DNA-PKcs module to regulate checkpoint responses. They emphasize notable similarities and distinct differences between G1-, G2- and S-phase checkpoint regulation that may guide DSB processing decisions.

Short communication: Canarium odontophyllum Miq. (dabai) stem bark arrested HCT 116 cell line at G0/G1 checkpoint

Canarium odontophyllum Miq. is an exotic plant which is native in Borneo and belong to the Burseraceae family. It contains phytochemicals such as saponin, terpenoid, flavonoid and phenolic compound with potential anticancer property. It has been found that the extract of this plant negatively affected colorectal cancer cells by stimulating apoptosis. To elucidate the apoptosis mechanism induced by the plant extract, this study evaluated the effect of C. odontophyllum stem bark acetone extract on cell cycle distribution of HCT 116 cell line using propidium iodide assay. For this purpose, IC50 of the acetone extract of C. odontophyllum was first determined by treating HCT 116 cells with the extract for 24, 48 and 72 hours. It was found that the acetone extract of C. odontophyllum inhibited proliferation of HCT 116 at IC50 value of 55.09 ± 18.29 µg/mL for 24 hours treatment, 37.81 ± 5.09 µg/mL for 48 hours treatment, and 114.9 ± 16.08 µg/mL following 72 hours treatment. Using IC50 value of 48 hours treatment, it was observed that C. odontophyllum acetone extract arrested the HCT 116 cells at G0/G1 checkpoint. Based on this result, it can be concluded that one of the apoptosis mechanisms induced by C. odontophyllum is by arresting cell cycle of HCT 116 cells at G0/G1 checkpoint. This finding warrants further investigation on how C. odontophyllum causes the cell cycle arrest and its potential to become anticancer agent.

Sustained CHK2 activity, but not ATM activity, is critical to maintain a G1 arrest after DNA damage in untransformed cells

Background The G1 checkpoint is a critical regulator of genomic stability in untransformed cells, preventing cell cycle progression after DNA damage. DNA double-strand breaks (DSBs) recruit and activate ATM, a kinase which in turn activates the CHK2 kinase to establish G1 arrest. While the onset of G1 arrest is well understood, the specific role that ATM and CHK2 play in regulating G1 checkpoint maintenance remains poorly characterized. Results Here we examine the impact of ATM and CHK2 activities on G1 checkpoint maintenance in untransformed cells after DNA damage caused by DSBs. We show that ATM becomes dispensable for G1 checkpoint maintenance as early as 1 h after DSB induction. In contrast, CHK2 kinase activity is necessary to maintain the G1 arrest, independently of ATM, ATR, and DNA-PKcs, implying that the G1 arrest is maintained in a lesion-independent manner. Sustained CHK2 activity is achieved through auto-activation and its acute inhibition enables cells to abrogate the G1-checkpoint and enter into S-phase. Accordingly, we show that CHK2 activity is lost in cells that recover from the G1 arrest, pointing to the involvement of a phosphatase with fast turnover. Conclusion Our data indicate that G1 checkpoint maintenance relies on CHK2 and that its negative regulation is crucial for G1 checkpoint recovery after DSB induction.

DNA damage checkpoint kinases in cancer

DNA damage response (DDR) pathway prevents high level endogenous and environmental DNA damage being replicated and passed on to the next generation of cells via an orchestrated and integrated network of cell cycle checkpoint signalling and DNA repair pathways. Depending on the type of damage, and where in the cell cycle it occurs different pathways are involved, with the ATM-CHK2-p53 pathway controlling the G1 checkpoint or ATR-CHK1-Wee1 pathway controlling the S and G2/M checkpoints. Loss of G1 checkpoint control is common in cancer through TP53, ATM mutations, Rb loss or cyclin E overexpression, providing a stronger rationale for targeting the S/G2 checkpoints. This review will focus on the ATM-CHK2-p53-p21 pathway and the ATR-CHK1-WEE1 pathway and ongoing efforts to target these pathways for patient benefit.

PDTM-38. HISTONE H3.3 SER31 PHOSPHORYLATION REGULATES CHROMOSOME SEGREGATION AND CELL CYCLE CHECKPOINT CONTROL AND IS IMPAIRED IN PEDIATRIC GLIOMAS

Diffuse intrinsic pontine glioma (DIPG) carries a signature mutation in histone H3.3 (K27M) that induces epigenetic reprogramming via suppressed Lys27 triple methylation (K27me3). H3.3 has a Ser at position 31, which is adjacent to the K27M DIPG mutation. H3.3 Ser31 is phosphorylated beginning in prophase. This phosphorylation is restricted to pericentromeric heterochromatin and becomes dephosphorylated in anaphase. Surprisingly, chromosome missegregation triggers Ser31 hyperphosphorylation on the lagging chromosome, and masking phosphoS31 prevents p53 accumulation; part of the G1 checkpoint response to aneuploidy. Whether the K27M mutation influences Ser31 phosphorylation, chromosome segregation or G1 checkpoint control is unknown. We show that K27M DIPG cells have reduced pericentromeric phosphoS31 and increased chromosome instability (CIN) compared to normal, diploid human cells. CRISPR-editing of K27M to M27K restored phosphoS31 to WT levels and dramatically decreased the rate of CIN. We also demonstrate that Chk1 is the H3.3 Ser31 kinase. S31A abolishes Ser31 phosphorylation by Chk1, while K27M reduces it by ~60% in vitro. Chk1 knockdown abolishes phosphoS31 in normal, diploid mitotic cells, and induces CIN. In normal, diploid cells, expression of S31A or K27M mutations increased chromosome missegregation, whereas cells expressing a phosphomimetic double mutant (K27M/S31E) divide normally. WT cells arrest following chromosome missegregation. Yet, normal cells expressing H3.3 K27M or S31A do not arrest - despite having wild-type p53. Finally, using RCAS/TVA, we expressed H3F3AS31A in the brains of BRAFV600E mice, and ~50% developed tumors, suggesting that loss of phosphoS31 alone is oncogenic. Importantly, H3.3 S31A-expressing cells are WT for K27me3. Our results demonstrate that expressing H3.3 K27M reduces the population of H3.3 that is phosphorylatable at Ser31, leading to chromosome missegregation and preventing G1 arrest – thus creating proliferating CIN cells. We propose that a single amino acid substitution drives oncogenesis of DIPG, at least impart, by triggering the evolution of complex karyotypes.