Transforming growth factor-beta is an endogenous radioresistance factor in the esophageal adenocarcinoma cell line OE-33.

Transforming growth factor (TGF)-beta has profound effects on epithelial cell differentiation and is capable of modulating the response to exposure to ionizing radiation. We recently reported that TGF-beta downregulates c-myc mRNA expression and inhibits the growth of OE-33 esophageal carcinoma cells in vitro. These studies investigate the role of TGF-beta in the in vitro radiation response of OE-33 and four other human esophageal cancer cell lines. TGF-beta enhanced radioresistance of OE-33 cells, but did not affect the radiosensitivity of either of the two other adenocarcinoma cell lines BIC1 and SEG1 or of squamous carcinomas KYSE and OE-21. The TGF-beta enhanced radioresistance phenotype was associated with induced G0/G1 cell cycle arrest and upregulation of the G1 cyclin-dependent kinase inhibitor p27kip1 as well as downregulation of c-myc protein expression. Comparison of the relative radiosensitivities of untreated cells suggested that OE-33 (SF2 = 0.71) cells were inherently more radioresistant than BIC1 or SEG1 cells (SF2 = 0.6 and 0.56, respectively). Conditioned medium obtained from unirradiated OE-33 cells enhanced radioresistance compared with fresh medium. This enhancement was abrogated by preincubation of conditioned medium with a neutralizing anti-TGF-beta antibody suggesting endogenous TGF-beta production by OE-33 cells. Enzyme-linked immunoabsorbent assays revealed that exposure to ionizing radiation increased TGF-beta production in all five cell lines. These results suggest that TGF-beta acts as an endogenous, radiation-inducible radioresistance factor in OE-33 esophageal carcinoma cells.

A simple method of producing low oxygen conditions with oxyrase for cultured cells exposed to radiation and tirapazamine.

Several methods of establishing low O(2) conditions have been used in studies on the response of cultured cells to radiation and other agents. These methods, eg, gassing culture vessels with O(2)-free nitrogen with or without carbon dioxide or placing high cell-density suspensions in sealed glass ampoules to consume O(2) in the ampules, can be technically demanding and have experimental limitations. We introduce a simple, versatile, and reliable method of producing low O(2) conditions without special equipment or changes in culture conditions unrelated to hypoxia. The method is based on the ability of Oxyrase (Oxyrase, Inc., Mansfield, OH), membrane fragments prepared from Enterococcus coli, to consume O(2) in solution and is confirmed in the present study by 2 analytical methods. The effects of low O(2) conditions induced by Oxyrase on cellular responses to radiation and treatment with the bioreductive agent tirapazamine (TPZ) were examined with Chinese hamster V79 and human glioma U373 cells. Measured by clonogenic and MTT assays, these cells were less sensitive to radiation but more sensitive to TPZ in treatment media containing native Oxyrase than in media containing heat-inactivated Oxyrase. In addition, Oxyrase treatment increased the basal activity of mitogen-activated protein kinase (ERK1/2) but suppressed its activation induced by radiation. The results suggest that this method might also be useful for other in vitro cancer biologic investigations requiring a low O(2) condition.

The effects of pentoxifylline on the survival of human glioma cells with continuous and intermittent stereotactic radiosurgery irradiation.

PURPOSE: In linac-based stereotactic radiosurgery, treatment is delivered intermittently via multiple individual small radiotherapy arcs. The time lapses between the individual arcs permit greater damage repair and increased tumor cell survival in comparison with continuous irradiation. Because pentoxifylline (PTX) has been reported to prevent radiation-induced cell cycle arrest at the G2/M checkpoint, where damage repair is critically linked to cell survival, we hypothesized that PTX would exert a favorable radiosensitization effect by reducing the recovery observed during intermittent stereotactic radiosurgery. METHODS AND MATERIALS: The human glioma cell line T98G was used to study the effects of continuous vs. intermittent irradiation with or without PTX. Cell cycle patterns were studied using flow cytometry. Clonogenic assays of single cells and spheroid outgrowth assays provided a quantitative measure of PTX-mediated radiosensitization. The PTX effect upon cells in low oxygen conditions was also studied in vitro after enzymatic oxygen scavenging. RESULTS: Flow Cytometry: T98G cells exposed to both continuous and intermittent irradiation exhibit similar arrest at the G2/M checkpoint. The addition of 2 mM PTX significantly reduced the radiation-induced G2/M block in both irradiation schemes. Clonogenic Assays: The same PTX concentration applied before a continuous dose of 12 Gy, two intermittent doses of 6 Gy, or three intermittent doses of 4 Gy, all given within a 1-h interval, consistently caused radiosensitization. The drug enhancement ratios for PTX were 1.5, 2.7, and 6.0 for the continuous and two different intermittent dose schedules, respectively. Adding PTX after irradiation yielded lower enhancement ratios than pre-irradiation application. A similar pattern was observed after total doses of 4, 6, 9, or 12 Gy, as well. In low oxygen conditions, PTX was seen to have the same effects as in normoxic conditions. Spheroid Outgrowth Assays: The in vitro PTX effects were replicated in the spheroid outgrowth assays. CONCLUSION: In human glioma cells, PTX abrogates the radiation-induced G2/M block observed after either continuous radiation exposure or intermittent exposures modeling clinical linac-based radiosurgery. The PTX-mediated reduction of the G2/M block translates into radiosensitization, most notably during intermittent exposures, and is presumably a consequence of diminished DNA damage repair at the G2/M checkpoint, though other contributing effects cannot be ruled out. The radiosensitization effect of PTX is sustained under low oxygen conditions. These results support consideration of the clinical evaluation of PTX to enhance the efficacy of linac-based radiosurgery involving intermittent irradiation through multiple arcs.