Detection of G1 cell cycle checkpoint integrity in tumor cells by radiolabeled FLT

Abstract

Purpose: Cell cycle checkpoint integrity plays an important role in both tumor progression and tumor response to therapy. We are developing methods to characterize checkpoint integrity in vivo. The objective of this study was to determine whether the thymidine analog [F-18] 3’-deoxy-3’-fluorothymidine (FLT), an agent being investigated for PET imaging of tumor cell proliferation, could detect differences in cell cycle checkpoint integrity.Materials and methods: We used two isogenic human lung adenocarcinoma cell lines derived from A549 cells. A549-E6 was transfected with a vector containing HPV type 16 E6 to abrogate the function of the tumor suppressor gene, p53, thereby eliminating the ability of radiation to inhibit movement of these cells through the cell cycle. A549-LXSN was transfected with a control vector. There were two types of experiments. In the first, plateau-phase cells were exposed to graded doses of radiation (2-10 Gy) and then released from plateau-phase by subculture. Twenty-four hours later, cells were cultured in the presence of [H-3]-FLT for 1 h and then harvested to measure FLT uptake. In the second experiment, exponentially growing cells were exposed to graded doses of radiation and then, at different times following irradiation (4-72 h), the cells were cultured in the presence of [H-3]-FLT for 1 h before being harvested to measure FLT uptake. For all experiments, the % S phase and TK1 activity were also determined.Results: In the plateau-phase experiments, there was a dose-dependent reduction in FLT uptake in A549-LXSN that corresponded to the reduction in %S phase cells and TK1 activity as increasing numbers of cells arrested in G1. In contrast, A549-E6 showed no decrease in FLT uptake, %S, or TK1 activity under these conditions as these cells failed to arrest in G1 following irradiation. For cells exposed under exponential growth conditions, A549-LXSN showed little change in FLT uptake following a 2-Gy exposure and only a small transient reduction in uptake following a 10-Gy exposure. For A549-E6, there was a small transient increase in FLT uptake following a 2-Gy exposure and a much larger increase following a 10-Gy exposure. As expected, FLT uptake was lowest in cells that arrested in G1. Surprisingly, FLT uptake was highest in cells that arrested in G2.Conclusion: FLT uptake following radiation exposure distinguishes cells with an intact G1 from those where the G1 checkpoint is compromised. Purpose: Cell cycle checkpoint integrity plays an important role in both tumor progression and tumor response to therapy. We are developing methods to characterize checkpoint integrity in vivo. The objective of this study was to determine whether the thymidine analog [F-18] 3’-deoxy-3’-fluorothymidine (FLT), an agent being investigated for PET imaging of tumor cell proliferation, could detect differences in cell cycle checkpoint integrity. Materials and methods: We used two isogenic human lung adenocarcinoma cell lines derived from A549 cells. A549-E6 was transfected with a vector containing HPV type 16 E6 to abrogate the function of the tumor suppressor gene, p53, thereby eliminating the ability of radiation to inhibit movement of these cells through the cell cycle. A549-LXSN was transfected with a control vector. There were two types of experiments. In the first, plateau-phase cells were exposed to graded doses of radiation (2-10 Gy) and then released from plateau-phase by subculture. Twenty-four hours later, cells were cultured in the presence of [H-3]-FLT for 1 h and then harvested to measure FLT uptake. In the second experiment, exponentially growing cells were exposed to graded doses of radiation and then, at different times following irradiation (4-72 h), the cells were cultured in the presence of [H-3]-FLT for 1 h before being harvested to measure FLT uptake. For all experiments, the % S phase and TK1 activity were also determined. Results: In the plateau-phase experiments, there was a dose-dependent reduction in FLT uptake in A549-LXSN that corresponded to the reduction in %S phase cells and TK1 activity as increasing numbers of cells arrested in G1. In contrast, A549-E6 showed no decrease in FLT uptake, %S, or TK1 activity under these conditions as these cells failed to arrest in G1 following irradiation. For cells exposed under exponential growth conditions, A549-LXSN showed little change in FLT uptake following a 2-Gy exposure and only a small transient reduction in uptake following a 10-Gy exposure. For A549-E6, there was a small transient increase in FLT uptake following a 2-Gy exposure and a much larger increase following a 10-Gy exposure. As expected, FLT uptake was lowest in cells that arrested in G1. Surprisingly, FLT uptake was highest in cells that arrested in G2. Conclusion: FLT uptake following radiation exposure distinguishes cells with an intact G1 from those where the G1 checkpoint is compromised.