Esophagitis is a major toxicity of radiation therapy for nonsmall‐cell lung cancer. Intraesophageal injection of manganese superoxide dismutase (MnSOD) plasmid/liposome complexes (1 mg of the pRK5‐MnSOD plasmid containing the human MnSOD transgene in a 0.15 ml volume of lipofectin) before irradiation was carried out to attempt to prevent irradiation esophagitis. In control noninjected male C3H/HeNsd mice, esophagitis was induced by single fraction 3,500 cGy irradiation. Histopathology at 4 days revealed vacuole formation in squamous lining cells, separation of the squamous layer from the underlying muscle layer, ulceration at 7 days, and dehydration and death by 30 days. MnSOD plasmid/liposome complex‐injected mice showed transcription of the human MnSOD transgene message in esophageal squamous lining cells by nested reverse transcriptase‐polymerase chain reaction (RT‐PCR) increased MnSOD biochemical activity 24 h after injection, decreased vacuole formation at day 4 (P < 0.001) after 3,500 cGy thoracic irradiation, and improved survival (P = 0.0009). In contrast, groups of mice receiving LacZ (bacterial β‐galactosidase gene) plasmid/liposome complexes or liposomes containing no DNA before 3,500 cGy irradiation showed an unaltered irradiation histopathology and decreased survival. Mice receiving intraesophageal MnSOD plasmid/liposomes followed 8 h later by human equivalent doses of Taxol (1.4 mg/kg) and carboplatin (2.5 mg/kg), then 15 h later 3,300 cGy irradiation, showed increased survival, compared with irradiated control or LacZ plasmid/liposome groups. Thus, overexpression of the human MnSOD transgene in the esophagus can prevent irradiation‐induced esophagitis in the mouse model. Radiat. Oncol. Invest. 7:204–217, 1999. © 1999 Wiley‐Liss, Inc.

To determine whether overexpression of the human MnSOD transgene protected 32D cl 3 hematopoietic progenitor cells from ionizing irradiation, 32D cl 3 cells were co‐electroporated with the pRK5 plasmid containing the human MnSOD transgene and SV2‐neo plasmid with G418‐resistant colonies selected. Two clones (1F2 and 2C6) were identified to overexpress the human MnSOD transgene by nested reverse transcriptase‐polymerase chain reaction (RT‐PCR) and increased biochemical activity. Measurement of irradiation‐induced damage was determined in cells removed from G418 for 1 week before irradiation. Irradiation survival curves, apoptosis tunnel assay, and Comet assay was performed. Cell cycle distribution was determined for each line at 0, 1, 3, 6, 24, and 48 hr after 500 cGy by fixing the cells in 70% ethanol, staining with propidium iodide, and analysis by flow cytometer. Biochemical MnSOD activity in U/mg protein was 2.6 for 32D cl 3 and significantly elevated to 8.4 and 6.6 (P < 0.001) U/mg protein for subclones 1F2 and 2C6, respectively. Irradiation survival curves demonstrated an increased shoulder on the irradiation survival curve for 1F2 and 2C6 cells with an n of 4.95 ± 0.48 (P = 0.042) and 4.95 ± 0.13 (P = 0.011), compared with 2.77 ± 0.20 for 32D cl 3. A higher percent of 32D cl 3 cells demonstrated apoptosis at 24 and 48 hr after 1,000 cGy irradiation, compared with 1F2 and 2C6 cells (at 24 hr, 29.37% ± 2.01% of 32D cl 3 cells were apoptotic compared with 5.21 ± 2.61 (P = 0.018) and 5.27 ± 2.58 (P = 0.004) for 1F2 and 2C6, respectively). Significantly more DNA strand breaks were detected by Comet assay in 32D cl 3 cells (Comet length at 600 cGy of 103.4 ± 50.3 units, compared with 69.7 ± 36.3 (P < 0.001) and 48.9 ± 27.5 (P < 0.001) for 1F2 and 2C6, respectively). In contrast, irradiation‐induced cell cycle arrest was similar between the cell lines with a G2/M phase arrest at 6 hr and a G1/S phase arrest at 24 and 48 hr after irradiation. While overexpression of MnSOD increases the shoulder on the irradiation survival curve of 32D cl 3 cells, decreases irradiation‐induced apoptosis, and DNA strand breaks by Comet assay, irradiation‐induced alterations in cell cycle distribution were not significantly altered. These 32D cl 3 subclonal lines overexpressing MnSOD provide a potentially valuable system with which to study the mechanism of irradiation‐induced cell cycle arrest separate from irradiation‐induced apoptosis. Radiat. Oncol. Invest. 7:331–342, 1999. © 1999 Wiley‐Liss, Inc.

Although radiotherapy and most cancer drugs target the proliferation of cancer cells, it is metastasis, the complex process by which cancer cells spread from the primary tumor to other tissues and organs of the body where they form new tumors, that leads to over 90% of all cancer deaths. Thus, there is an urgent need for anti-metastasis strategies alongside chemotherapy and radiotherapy. An important step in the metastatic cascade is migration. It is the first step in metastasis via local invasion. Here we address the question whether ionizing radiation and/or chemotherapy might inadvertently promote metastasis and/or invasiveness by enhancing cell migration. We used a standard laboratory irradiator, Faxitron CellRad, to irradiate both non-cancer (HCN2 neurons) and cancer cells (T98G glioblastoma) with 2 Gy, 10 Gy and 20 Gy of X-rays. Paclitaxel (5 μM) was used for chemotherapy. We then measured the attachment and migration of the cells using an electric cell substrate impedance sensing device. Both the irradiated HCN2 cells and T98G cells showed significantly (p < 0.01) enhanced migration compared to non-irradiated cells, within the first 20–40 h following irradiation with 20 Gy. Our results suggest that cell migration should be a therapeutic target in anti-metastasis/anti-invasion strategies for improved radiotherapy and chemotherapy outcomes.

Although radiotherapy and most cancer drugs target the proliferation of cancer cells, it is metastasis, the complex process by which cancer cells spread from the primary tumor to other tissues and organs of the body where they form new tumors, that leads to over 90% of all cancer deaths. Thus, there is an urgent need for anti-metastasis strategies alongside chemotherapy and radiotherapy. An important step in the metastatic cascade is migration. It is the first step in metastasis via local invasion. Here we address the question whether ionizing radiation and/or chemotherapy might inadvertently promote metastasis and/or invasiveness by enhancing cell migration. We used a standard laboratory irradiator, Faxitron CellRad, to irradiate both non-cancer (HCN2 neurons) and cancer cells (T98G glioblastoma) with 2 Gy, 10 Gy and 20 Gy of X-rays. Paclitaxel (5 μM) was used for chemotherapy. We then measured the attachment and migration of the cells using an electric cell substrate impedance sensing device. Both the irradiated HCN2 cells and T98G cells showed significantly (p < 0.01) enhanced migration compared to non-irradiated cells, within the first 20 to 40 hours following irradiation with 20 Gy. Our results suggest that cell migration should be a therapeutic target in anti-metastasis/anti-invasion strategies for improved radiotherapy and chemotherapy outcomes.