Microcolony survival assay for jejunal crypt cells exposed to radiation alone and combined with cancer chemotherapeutic agents--methodological problems

Abstract

The effect of radiation alone or in combination with cyclophosphamide (CTX), methotrexate (MTX) and 5-fluorouracil (5-FU) on the jejunal crypt cells in C3D2F1/Bom mice was studied using the microcolony survival assay. In determination of survival curves, two sections per mouse and six mice per datum point were used in order to obtain a constant coefficient of variance of D0 (approximately 5 per cent). When CTX (250 mg/kg), MTX (150mg/kg), and 5-FU (150 mg/kg) were injected intraperitoneally 15 min before irradiation, the regeneration time for the surviving crypts increased. Thus, following radiation alone the regeneration time was 90 hours and when combined with the drugs, 96, 102, and 120 hours, respectively. At these hours the crypts were of an equivalent size. Scoring the crypt number at the regeneration times was found to be necessary, since the use of a constant assay time underestimated the crypt survival following the drug-radiation combinations. Neither was a constant assay time followed by a correction for different crypt sizes found sufficient to replace the use of different regeneration times. All three drugs significantly enhanced the radiation effect without changing the D0. The DEF values for CTX, MTX, and 5-FU were 1 . 05, 1 . 08 and 1 . 13, respectively.