Increase in the fraction of necrotic, not apoptotic, cells in SiHa xenograft tumours shortly after irradiation.
OBJECTIVE
Approximately 18% of the cells recovered by rapid mechanical dissociation of SiHa xenograft tumours contain large numbers of DNA strand breaks. The number of damaged cells increases to 30-40% 4-6 h after exposure to 5 or 15 Gy, returning to normal levels by 12 h. This observation is reminiscent of the rate of production of apoptotic cells in other murine and human xenograft tumours. The nature of this damage, rate of development and relation to cell proliferation rate were therefore examined in detail.


METHODS
SiHa human cervical carcinoma cells were grown as xenograft tumours in SCID mice. Single-cell suspensions were prepared as a function of time after irradiation of the mouse and examined for DNA damage using 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 irradiated SiHa tumours using an end-labelling assay, electron microscopy, or histological examination of thin sections. Instead, xenograft cells exhibiting extensive DNA damage in the comet assay were predominantly necrotic cells. The increase in the proportion of heavily damaged cells 4-6 h after irradiation could be the result of an interplay between several factors including loss of viable cells and change in production or loss of necrotic cells. Analysis of the progression of BrdUrd-labelled cells confirmed that while 35% of cells from untreated SiHa tumours had divided and entered G1 phase by 6 h after BrdUrd injection, none of the labelled cells from tumours exposed to 5 or 15 Gy had progressed to G1.


CONCLUSIONS
The increase in the percentage of SiHa tumour cells with extensive DNA damage 4-6 h after irradiation is attributable to necrosis, not apoptosis. Cell cycle progression and cell loss are likely to influence the kinetics of appearance of both apoptotic and necrotic cells in irradiated tumours.
Olive
P L
PL
Medical Biophysics Department, British Columbia Cancer Research Centre, Vancouver, Canada.
Vikse
C M
CM
Vanderbyl
S
S
D000818:Animals; D000963:Antimetabolites; D017209:Apoptosis; D001973:Bromodeoxyuridine; D002277:Carcinoma / Q000473:pathology* / Q000532:radiotherapy; D002453:Cell Cycle; D002455:Cell Division / Q000528:radiation effects; D002470:Cell Survival / Q000528:radiation effects; D004249:DNA Damage; D004273:DNA, Neoplasm / Q000235:genetics / Q000528:radiation effects; D005434:Flow Cytometry; D016193:G1 Phase; D006801:Humans; D051379:Mice; D016513:Mice, SCID; D008854:Microscopy, Electron; D009336:Necrosis; D009368:Neoplasm Transplantation; D011879:Radiotherapy Dosage; D012878:Skin Neoplasms / Q000473:pathology* / Q000532:radiotherapy; D014183:Transplantation, Heterologous; D014407:Tumor Cells, Cultured
D000963:Antimetabolites; D004273:DNA, Neoplasm; D001973:Bromodeoxyuridine