Increase in the fraction of necrotic, not apoptotic, cells in SiHa xenograft tumours shortly after irradiation

Background and purpose: Approximately 18% of the cells recovered by rapid m echanical dissociation of SiHa xenograft tumours contain large numbers of D NA strand breaks. The number of damaged cells increases to 30-40% 4-6 h aft er exposure to 5 or 15 Gy, returning to normal levels by 12 h. This observa tion is reminiscent of the rate of production of apoptotic cells in other m urine and human xenograft tumours. The nature of this damage, rate of devel opment and relation to cell proliferation rate were therefore examined in d etail. Materials and methods: SiHa human cervical carcinoma cells were grown as xe nograft tumours in SCID mice. Single-cell suspensions were prepared as a fu nction of time after irradiation of the mouse and examined for DNA damage u sing the alkaline comet assay. Cell cycle progression was measured by flow cytometry evaluation of anti-bromodeoxyuridine-labelled tumour cells. Results: Significant numbers of apoptotic cells could not be detected in ir radiated SiHa tumours using an end-labelling assay, electron microscopy, or histological examination of thin sections. Instead, xenograft cells exhibi ting extensive DNA damage in the comet assay were predominantly necrotic ce lls. The increase in the proportion of heavily damaged cells 4-6 h after ir radiation could be the result of an interplay between several factors inclu ding loss of viable cells and change in production or loss of necrotic cell s. Analysis of the progression of BrdUrd-labelled cells confirmed that whil e 35% of cells from untreated SiHa tumours had divided and entered G(1) pha se by 6 h after BrdUrd injection, none of the labelled cells from tumours e xposed to 5 or 15 Gy had progressed to G(1). Conclusions: The increase in the percentage of SiHa tumour cells with exten sive DNA damage 4-6 h after irradiation is attributable to necrosis, not ap optosis. Cell cycle progression and cell loss are likely to influence the k inetics of appearance of both apoptotic and necrotic cells in irradiated tu mours. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.