Radiobiologic significance of apoptosis and micronucleation in quiescent cells within solid tumors following gamma-ray irradiation.
OBJECTIVE
To determine the frequency of apoptosis in quiescent (Q) cells within solid tumors following gamma-ray irradiation, using four different tumor cell lines. In addition, to assess the significance of detecting apoptosis in these cell lines.


METHODS
C3H/He mice bearing SCC VII or FM3A tumors, Balb/c mice bearing EMT6/KU tumors, and C57BL mice bearing EL4 tumors received 5-bromo-2'-deoxyuridine (BrdU) continuously for 5 days via implanted mini-osmotic pumps to label all proliferating (P) cells. The mice then received gamma-ray irradiation at a dose of 4--25 Gy while alive or after tumor clamping. Immediately after irradiation, the tumors were excised, minced, and trypsinized. The tumor cell suspensions thus obtained were incubated with cytochalasin-B (a cytokinesis blocker), and the micronucleus (MN) frequency in cells without BrdU labeling (= Q cells) was determined using immunofluorescence staining for BrdU. Meanwhile, 6 hours after irradiation, tumor cell suspensions obtained in the same manner were fixed. The apoptosis frequency in Q cells was also determined with immunofluorescence staining for BrdU. The MN and apoptosis frequency in total (P + Q) tumor cells were determined from the tumors that were not pretreated with BrdU.


RESULTS
In total cells, SCC VII, FM3A, and EMT6/KU cells showed reasonable relationships between MN frequency and surviving fraction (SF). However, fewer micronuclei were induced in EL4 cells than the other cell lines. In contrast, a comparatively close relationship between apoptosis frequency and SF was found in total cells of EL4 cell line. Less apoptosis was observed in the other cell lines. Quiescent tumor cells exhibited significantly lower values of MN and apoptosis frequency probably due to their large hypoxic fraction, similar to total tumor cells on clamped irradiation.


CONCLUSIONS
gamma-ray irradiation induced MN formation in SCC VII, FM3A, and EMT6/KU tumor cells, and the apoptosis was marked in EL4 cells compared with the other cell lines. Our method for detecting the Q cell response to gamma-ray irradiation using P cell labeling with BrdU and the MN frequency assay was also applicable to apoptosis detection assay.
Masunaga
S
S
Radiation Oncology Research Laboratory, Research Reactor Institute, Kyoto University, Osaka, Japan. smasuna@rri.kyoto-u.ac.jp
Ono
K
K
Suzuki
M
M
Kinashi
Y
Y
Takagaki
M
M
D000818:Animals; D000911:Antibodies, Monoclonal; D017209:Apoptosis / Q000502:physiology*; D001973:Bromodeoxyuridine; D015687:Cell Hypoxia; D002470:Cell Survival / Q000528:radiation effects*; D004307:Dose-Response Relationship, Radiation; D020287:In Situ Nick-End Labeling; D051379:Mice; D008807:Mice, Inbred BALB C; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D048629:Micronuclei, Chromosome-Defective; D015162:Micronucleus Tests; D011853:Radiobiology; D014407:Tumor Cells, Cultured / Q000528:radiation effects
D000911:Antibodies, Monoclonal; D001973:Bromodeoxyuridine