sepharose and Carcinoma--Small-Cell

Among the techniques available for the measurement of the induction and rejoining of DNA double-strand breaks (DSBs), pulsed-field gel electrophoresis appears to have the greatest potential to improve the sensitivity limits to study these lesions in the dose range closest to that used in cell survival experiments. Encapsulating the cells in agarose during the experimental procedure allows the accurate and reproducible measurement of rejoining kinetics with a very minimal time delay immediately after irradiation. The method allows direct comparison of the amount of initial DNA damage sustained with repair kinetics in experiments designed to elucidate the mechanisms underlying differences in radiosensitivity between cell lines, together with analysis of the effect of different radiation qualities. The sensitivity limits of the method are 1 Gy for the double-strand break induction experiments and 10 Gy for rejoining experiments. Under selected conditions, no significant degradation of DNA had been observed in rodent cell lines during repair incubation up to 17 h in either irradiated cells or unirradiated controls (background levels for neutron experiments, 2.2 +/- 0.3% at Time 0 compared to 2.3 +/- 0.5% after 17 h of incubation; background levels for X-ray experiments, 2.3 +/- 0.6% at Time 0 and 3.7 +/- 1.1% after 17 h of incubation). In preliminary experiments with the A549 human oat cell carcinoma cell line, DNA DSB background levels remained constant in unirradiated controls up to 4 h in the range reported for the rodent cell line.

Topics: Animals; Carcinoma, Small Cell; Cell Line; Cricetinae; DNA; DNA Damage; DNA Repair; DNA, Neoplasm; Electrophoresis, Gel, Pulsed-Field; Humans; Sepharose; Tumor Cells, Cultured

Twenty-seven specimens for in vitro agarose clonogenicity testing were obtained from 25 patients with small-cell carcinoma of the lung (SCCL). The specimens were obtained from bone marrows, pleural effusions, lymph nodes, and liver biopsies. Colony formation was seen in 14 of 15 specimens that were histologically involved with SCCL, but no colony growth was seen in the 12 patient specimens without histocytopathological evidence of SCCL, including seven bone marrow specimens. Cytological examination of the agarose colonies confirmed their SCCL origin. Colonies reached sizes of 50 to 1000 cells in 7 to 10 days, indicating an in vitro doubling time of less than 24 hr, remarkably shorter than the population doubling times measured in patients. None of the 100 clones picked from these specimens demonstrated the ability to continuously replicate in vitro. These results show an excellent correlation between agarose colony formation and histological tumor involvement and a more rapid in vitro doubling time than that seen in vivo and demonstrate that standard tissue culture conditions do not allow demonstration of a self-renewing stem cell in fresh tumor specimens of SCCL.

Topics: Carcinoma, Small Cell; Cell Division; Cells, Cultured; Clone Cells; Culture Media; Humans; Lung Neoplasms; Neoplasm Metastasis; Sepharose