G2/M Checkpoint Abrogation With Selective Inhibitors Results in Increased Chromatid Breaks and Radiosensitization of 82-6 hTERT and RPE Human Cells

Abstract

While technological advances in radiation oncology have led to a more precise delivery of radiation dose and a decreased risk of side effects, there is still a need to better understand the mechanisms underlying DNA damage response (DDR) at the DNA and cytogenetic levels, and to overcome tumor resistance. To maintain genomic stability, cells have developed sophisticated signaling pathways enabling cell cycle arrest to facilitate DNA repair via the DDR-related kinases and their downstream targets, so that DNA damage or DNA replication stress induced by genotoxic therapies can be resolved. ATM, ATR, and Chk1 kinases are key mediators in DDR activation and crucial factors in treatment resistance. It is of importance, therefore, as an alternative to the conventional clonogenic assay, to establish a cytogenetic assay enabling reliable and time-efficient results in evaluating the potency of DDR inhibitors for radiosensitization. Toward this goal, the present study aims at the development and optimization of a chromosomal radiosensitivity assay using the DDR and G2-checkpoint inhibitors as a novel modification compared to the classical G2-assay. Also, it aims at investigating the strengths of this assay for rapid radiosensitivity assessments in cultured cells, and potentially, in tumor cells obtained from biopsies. Specifically, exponentially growing RPE and 82-6 hTERT human cells are irradiated during the G2/M-phase transition in the presence or absence of Caffeine, VE-821, and UCN-1 inhibitors of ATM/ATR, ATR, and Chk1, respectively, and the induced chromatid breaks are used to evaluate cell radiosensitivity and their potency for radiosensitization. The increased yield of chromatid breaks in the presence of DDR inhibitors, which underpins radiosensitization, is similar to that observed in cells from highly radiosensitive AT-patients, and is considered here as 100% radiosensitive internal control. The results highlight the potential of our modified G2-assay using VE-821 to evaluate cell radiosensitivity, the efficacy of DDR inhibitors in radiosensitization, and reinforce the concept that ATM, ATR, and Chk1 represent attractive anticancer drug targets in radiation oncology.

Replication Stress: A Review of Novel Targets to Enhance Radiosensitivity-From Bench to Clinic

“…When DDR is activated, it temporarily stops the cell cycle to provide more time for repair, or if the damage is too severe, induces apoptosis. Eliminating the radiation-induced G2/M arrest or forcing damage cells to enter into mitosis both sensitizes cancer cells to radiation treatment (45,46). This cellcycle-dependent radiosensitization mechanism provides potential directions for further research into radiosensitizers.…”

A specific dispiropiperazine derivative that arrests cell cycle, induces apoptosis, necrosis and DNA damage

“…The anti-proliferative properties of SPOPP-3 (1) has important implications in cancer therapy. Synthetic lethality is a novel approach in cancer treatment 4,5 . Since SPOPP-3 (1) is a potent inducer of G2/M arrest, it has the potential to be used in combination with G2/M checkpoint inhibitors to trigger mitotic catastrophe which is currently viewed as a favorable treatment strategy to enhance cell death either via apoptosis, necrosis or autophagy [1][2][3][4][5] .…”

“…Synthetic lethality is a novel approach in cancer treatment 4,5 . Since SPOPP-3 (1) is a potent inducer of G2/M arrest, it has the potential to be used in combination with G2/M checkpoint inhibitors to trigger mitotic catastrophe which is currently viewed as a favorable treatment strategy to enhance cell death either via apoptosis, necrosis or autophagy [1][2][3][4][5] . Particularly, in most of melanoma cases where G1/S transition mediated by the cyclin-CDK4 pathway is defective and has increased dependence on the G2/M checkpoint to induce cell cycle arrest when exposed to DNA damage, SPOPP-3 (1) may have an added advantage in combination with G2/M inhibitors 4 .…”

“…The use of chemicals and radiation to induce DNA damage is the most commonly used method for cancer therapy. Recently, there is an increased interest in manipulating the cell cycle to induce mitotic catastrophe as a novel anti-cancer therapeutic strategy [1][2][3][4][5] . Mitotic catastrophe is characterized by cells which would normally be arrested in G2/M phase due to damage in DNA or mitotic spindle but falsely proceed to mitosis due to defective cell cycle checkpoints 6 .…”

“…This strategy relies on the use of DNA damaging agents or radiation in combination with cell cycle checkpoint inhibitors. Indeed, several G2/M phase checkpoint inhibitors [1][2][3][4][5] , including irinotecan, a currently used chemotherapeutic drug for metastatic colorectal cancer, have shown potential to sensitize tumor cells to ionizing radiation. As such, the discovery of new compounds which cause G2/M cell cycle arrest remains an important area of cancer research [8][9][10] .…”