Apoptosis induction and micronuclei formation were compared following cytotoxic treatments in two rat glioma differing in p53 integrity. In vitro, micronuclei emergence but not apoptosis was linked to the p53 mutated status. In vivo, micronuclei assays were more sensitive to evaluate DNA damage induced by chemotherapy in a p53-mutated solid tumour. (Mow et al, 2001). The product of the tumour-suppressor gene p53 is a key mediator in this process (Soussi, 2000). In normal cells, wild-type p53 either arrests cell proliferation until the genetic damage is repaired or forces the cell to commit apoptosis (Cadwell and Zambetti, 2001). In cancer cells, where p53 alleles are often mutated, decrease in apoptosis can be compensated by the process of mitotic catastrophe, a form of cell death resulting from abnormal mitosis and leading to the formation of cells with multiple micronuclei (MN) (Roninson et al, 2001). This study aimed to compare induction of apoptosis and MN formation after chemo-or radiotherapy, in vitro in two rat glioma cell lines differing in p53 integrity, the 9L expressing a mutated p53 gene and the C6 the wild-type gene, and in vivo on established 9L solid tumours.
MATERIALS AND METHODS
Animals and cell linesAll experiments on Fischer 344 rats have been carried out with local ethical committee approval and met the standards required by the UKCCCR guidelines (Workman et al, 1998). The 9L gliosarcoma cell line and the C6 glioblastoma cell line were maintained in complete medium, RPMI 1640 supplemented with 10% foetal calf serum, 1% L-glutamine, 1% sodium pyruvate, 1% nonessential amino acids, 100 IU ml Ă1 penicillin and 100 mg ml Ă1 streptomycin.
In vitro and in vivo radio-or chemotherapyFor in vitro treatments, 9L and C6 cells were g-irradiated (80 Gy, 137 Cs irradiator) or treated for 2 h with chemotherapeutic drugs and recultured during 24 or 72 h for apoptosis tests or 4 h for MN assays. For in vivo treatments, tumour-bearing rats (10 5 9L s.c. at day 0) received i.p. injections of cisplatin (1 mg kg Ă1 ), or were irradiated locally at the tumour site (20 Gy) at days 4, 11 and 18 and were killed the day after. The tumours were then dissected and prepared distinctly for apoptosis or MN assays.
Apoptosis assaysAnalysis of caspase 3 activity Caspase-3 activation was measured in vitro by the cleavage of a specific fluorogenic substrate (Ac-DEVD-AMC, BD Biosciences, Erembodegem, Belgium). Briefly, 24 or 72 h after treatment, cells were lysed and incubated first in protease buffer and then for 2 h at 371C with Ac-DEVD-AMC substrate (1 mg ml Ă1 ). The enzyme-catalysed release of fluorescent AMC was measured, by referring to a standard curve, with a fluorimeter, at 380 nm excitation and 440 nm emission wavelengths.Measurement of phosphatidylserine translocation using AnnexinV -FITC Phosphatidylserine outer translocation on treated cells was detected by flow cytometry (BD FACScan and CellQuest software) using AnnexinV -FITC binding (1 mg ml Ă1 ) (BD Biosciences) and PI (2 mg ml Ă1 ) counterstaining.TUNEL a...