VIII-Ⅱ-3. Original Research

OR1

Effect of Tirapazamine and Mild Temperature Hyperthermia on the Recovery
from Radiation-Induced Damage in Pimonidazole-Unlabeled Quiescent Tumor Cells

S. Masunaga, H. Tanaka, Y. Sakurai, M. Suzuki, K. Tano,
N. Kondo, M. Narabayashi, A. Maruhashi and K. Ono
Research Reactor Institute, Kyoto University
BACKGROUNDS AND PURPOSES: Human solid
tumors are thought to contain moderately large fractionsof quiescent (Q) tumor cells, which are not involved inthe cell cycle and have stopped dividing, but which are asviable as established experimental animal tumor lines.
The presence of Q cells is probably due, at least in part,to hypoxia and the depletion of nutrition in the tumor
core as a consequence of poor vascular supply. As aresult, with the exception of non-viable Q cells at thevery edge of the necrotic rim where there isdiffusion-limited hypoxia, Q cells are viable andclonogenic but have ceased dividing.
A few years ago, the universal detection of hypoxic
cells in both tissues and cell cultures became possibleusing pimonidazole, a substituted 2-nitroimidazole, and amouse IgG1 monoclonal antibody (MAb1) to stable covalent adducts formed through reductive activation ofpimonidazole in hypoxic cells. Here, we tried to selectively detect the response of the pimonidazole-unlabeledand probably oxygenated cell fraction of the Q cell population. To achieve this we combined our method forselectively detecting the response of Q cells in solid tumors with the method for detecting cell and tissue hypoxia using pimonidazole and MAb1 to pimonidazole.
The development of bioreductive agents that are particularly toxic to hypoxic cells is considered a promising approach to solving the problem of radio-resistant tumor
hypoxia in cancer radiotherapy. Tirapazamine (TPZ), a
lead compound in the development of bioreductive hypoxic cytotoxins, in combination with radiation has beenshown to be very useful for controlling solid tumors as awhole, especially for controlling Q tumor cell populations that are rich in hypoxic region. Mild temperature
hyperthermia (MTH) was reported to increase the response of tumors to radiation by improving oxygenation
through an increase in tumor blood flow. Further, MTH
was also shown to enhance the tumor response, especiallyof the intratumor Q cell population, to TPZ.
In the present study, the radio-sensitivity of the pimonidazole-unlabeled cell fraction of the Q cell population after cobalt-60 γ-ray irradiation followed with orwithout post-irradiation TPZ administration or MTH, wasdetermined, to examine the effect of TPZ and MTH on
the repair of radiation-induced damage in pimonidazole-unlabeled Q tumor cell population.
MATERIALS AND METHODS: Labeling of proliferating (P) cells in C57BL/6J mice bearing EL4 tumors
was achieved by continuous administration of
5-bromo-2-deoxyuridine (BrdU). Tumors were irradiatedwith γ-rays at 1 h after the administration of pimonidazole followed by TPZ treatment or MTH. Twenty-fourhours later, assessment of the responses of Q and total (=P + Q) cells were based on the frequencies of micronucleation and apoptosis using immunofluorescence staining for BrdU. The response of the pimonidazole-unlabeled tumor cell fractions was assessed bymeans of apoptosis frequency using immunofluorescencestaining for pimonidazole.
RESULTS: With γ-rays only, the pimonidazole- unlabeled cell fraction showed significantly enhanced radio-sensitivity compared with the whole cell fraction
more remarkably in Q cells than total cells. However, asignificantly greater decrease in radio-sensitivity in thepimonidazole-unlabeled than the whole cell fraction,
evaluated using a delayed assay, was more clearly observed in Q cells than total cells. Post-irradiation MTH
more remarkably repressed the decrease in radio-sensitivity in the Q cell than the total cells.
Post-irradiation TPZ administration produced a large radio-sensitizing effect on both total and Q cells, especiallyon Q cells. On the other hand, in pimonidazole-unlabeledcell fractions in both total and Q cells, TPZ suppressed
the reduction in sensitivity due to delayed assay much
more efficiently than MTH, whereas no radio-sensitizing
effect was produced.
CONCLUSION: Not only through suppressing therecovery from radiation-induced damage but also throughradio-sensitizing effect, post-irradiation TPZ administration is very useful for repressing the increase in the difference in radio-sensitivity due to the delayed assay not
only between total and Q tumor cells but also between
the pimonidazole-unlabeled and the whole cell fractions
within the total and Q tumor cells [1].

REFERENCES:[1] S. Masunaga et al., J. Cancer Ther., 4 (2013) 521-528.

ピモニダゾール非標識酸素化休止期腫瘍細胞分画における放射線損傷からの独自研究
回復に対するティラパザミン処置と低温度温熱処置の影響（京大・原子炉）増永慎一郎、田中浩基、櫻井良憲、鈴木実、田野恵三、近藤夏子、楢林正流、
丸橋 晃、小野公二