Role of cell cycle in epidermal growth factor receptor inhibitor-mediated radiosensitization

Abstract

Epidermal growth factor receptor (EGFR) inhibitors are increasingly used in combination with radiotherapy in the treatment of various EGFR-overexpressing cancers. However, little is known about the effects of cell cycle status on EGFR inhibitor-mediated radiosensitization. Using EGFR-overexpressing A431 and UMSCC-1 cells in culture, we found that radiation activated the EGFR and extracellular signal-regulated kinase pathways in quiescent cells, leading to progression of cells from G(1) to S, but this activation and progression did not occur in proliferating cells. Inhibition of this activation blocked S-phase progression and protected quiescent cells from radiation-induced death. To determine if these effects were caused by EGFR expression, we transfected Chinese hamster ovary (CHO) cells, which lack EGFR expression, with EGFR expression vector. EGFR expressed in CHO cells also became activated in quiescent cells but not in proliferating cells after irradiation. Moreover, quiescent cells expressing EGFR underwent increased radiation-induced clonogenic death compared with both proliferating CHO cells expressing EGFR and quiescent wild-type CHO cells. Our data show that radiation-induced enhancement of cell death in quiescent cells involves activation of the EGFR and extracellular signal-regulated kinase pathways. Furthermore, they suggest that EGFR inhibitors may protect quiescent tumor cells, whereas radiosensitization of proliferating cells may be caused by downstream effects such as cell cycle redistribution. These findings emphasize the need for careful scheduling of treatment with the combination of EGFR inhibitors and radiation and suggest that EGFR inhibitors might best be given after radiation in order to optimize clinical outcome.

Figure 1. Effect of cell culture condition on the radiation sensitivity of A431 and UMSCC-1 cells

A). Proliferating and quiescent A431 and UMSCC-1 cells were exposed to various doses of radiation and plated for clonogenic survival assays. Quiescent cells were more sensitive to radiation than proliferating cells (ratio of surviving fractions = 2.70 at 4 Gy). B). Cells were treated with 4 Gy and harvested at various time points after radiation. Levels of pEGFR, EGFR, pERK1/2, ERK1/2, γH2ax and GAPDH proteins were measured in the cell lysates. Immediate activation of EGFR and ERK (by 5 min) was induced in quiescent cells, but not in proliferating cells. DNA damage in proliferating cells was repaired by 2 hours, but remained unrepaired in quiescent cells.

Figure 2. Effect of radiation on cell cycle in A431 quiescent cells

A). Cell cycle distribution was analyzed by flow cytometry as described in Materials and Methods. An increase in apoptotic cells was observed in quiescent cells as compared to proliferating cells at 24 hours after radiation (4 Gy). Radiation induced substantial movement of cells from G₁ to S-phase in quiescent cells, but not in proliferating cells. B). Quiescent A431 cells were treated with 10 μM U0126 (1h), 10 ng/ml EGF (30 min) and 3 μM Erlotinib (2h) and exposed to 4 Gy radiation, and harvested 2h later. BrdUrd incorporation was performed as described in Materials and Methods. U0126 and erlotinib each blocked the progression of quiescent cells to S-phase in response to radiation. EGF stimulation induced quiescent cell progression to S-phase in response to radiation.

Figure 3. Effect of EGFR inhibition by erlotinib, stimulation by EGF, or ERK inhibition by U0126 on radiation sensitivity of A431 cells

A). Quiescent and B). Proliferating cells were pre-treated with erlotinib (3 μM for 1h), EGF (10 ng/ml for 30 min) or U0126 (10 μM for 1h) and exposed to various doses of radiation. Cells were immediately plated for colony formation. EGF pretreatment sensitized cells to radiation (enhancement ratio = 1.3 ± 0.01), and both erlotinib and U0126 pretreatment protected cells from radiation-induced cell death (enhancement ratio = 0.53 ± 0.25; p<0.002 and 0.62 ±0.14; p<0.005, respectively). C). Using the same conditions of drug treatment, cells were harvested at various time points after 4 Gy radiation. Immunoblotting was performed for pEGFR, EGFR, pERK1/2, ERK1/2 and GAPDH. D). Cells were harvested at 1, 2 and 3h after U0126 treatment and immunoblotted for pERK1/2 and ERK1/2.

Figure 4. Effect of EGFR expression on radiation sensitivity in proliferating and quiescent CHO cells

Proliferating and quiescent CHO cells were transfected with A) empty vector plasmids or B) wild-type EGFR plasmids and exposed to various doses of radiation and plated for clonogenic survival assays. Those transfected with empty vector plasmids showed no difference in clonogenic survival at any dose of radiation. In CHO cells transfected with EGFR, clonogenic survival revealed increased radiation sensitivity of quiescent cells compared to proliferating cells (enhancement ratio = 1.5 ± 0.2, p <0.001). C) Proliferating and quiescent cells were transfected with empty vector plasmids or D) wild type EGFR plasmids and treated with 4Gy radiation. Cells were harvested at various time points and immunoblotted for pEGFR and pERK. Ectopic expression of EGFR resulted in EGFR activation after radiation only in quiescent cells, and not in proliferating cells.