Masunaga Shin-ichiro, Uto Yoshihiro, Nagasawa Hideko, Hori Hitoshi, Nagata Kenji, Suzuki Minoru, Kinashi Yuko, Ono Koji
Radiation Oncology Research Laboratory, Research Reactor Institute, Kyoto University, 2-1010, Asashiro-nishi, Kumatori-cho, Sennan-gun, Osaka 590-0494, Japan.
Anticancer Res. 2006 Mar-Apr;26(2A):1261-70.
Intratumor quiescent (Q) cells and p53-mutated tumor cells are more difficult to control than intratumor proliferating (P) cells and p53 wild-type tumor cells, respectively. The usefulness of 3 hypoxic cell radio-sensitizers was compared in terms of a radio-sensitizing effect under aerobic and hypoxic conditions and a repair-inhibiting effect following irradiation on both Q and total (P + Q) cell populations in solid tumors. The dependency of these effects on the p53 status of tumor cells was also examined using tumor cell lines with identical genetic backgrounds except for their p53 status.
Human head and neck squamous cell carcinoma cells transfected with mutant TP53 (SAS/mp53) or with neo vector as a control (SAS/neo) were inoculated subcutaneously into both the hind legs of Balb/cA nude mice. The nude mice bearing the tumors and C3H/He mice bearing SCC VII tumors received 5-bromo-2'-deoxyuridine (BrdU) continuously to label all the P cells in the tumors. Tumor-bearing mice received gamma-ray irradiation while alive or following tumor clamping after being administered no drug, nimorazole, SR-2514 or misonidazole, or received no drug, nimorazole, SR-2514 or misonidazole straight after gamma-ray irradiation. For the group irradiated after receiving the drug, the tumors were excised immediately following irradiation, while for the group irradiated before receiving the drug, the tumors were excised 24 h after irradiation. The excised tumors were minced and trypsinized. The tumor cell suspensions thus obtained were incubated with cytochalasin-B (a cytokinesis blocker), and the micronucleus (MN) frequency in the cells without BrdU labelling (= quiescent (Q) cells) was determined using immunofluorescence staining for BrdU. Meanwhile, the MN frequency in the total tumor cell population was determined from the tumors that had not been pretreated with BrdU. The clonogenic cell survival was also determined in the mice given no BrdU.
Both the radio-sensitizing effects under aerobic and hypoxic conditions and the repair-inhibiting effects following gamma-ray irradiation increased in the following order: nimorazole < SR-2514 < misonidazole in both total and Q cells in these 3 tumors. Both effects were more marked in the Q cells and p53-mutated tumors than in the total cells and p53-wild tumors, respectively.
In terms of controlling radio-resistant Q tumor cells and p53-mutated tumor cells, the combination of radio-sensitizers and conventional radiotherapy is promising both for radio-sensitization and for repair-inhibition, but further study of the toxicity to normal tissues is needed.
肿瘤内静止（Q）细胞和p53突变的肿瘤细胞分别比肿瘤内增殖（P）细胞和p53野生型肿瘤细胞更难控制。比较了3种乏氧细胞放射增敏剂在有氧和乏氧条件下的放射增敏作用以及对实体瘤中Q细胞群和总（P + Q）细胞群照射后的修复抑制作用。还使用了除p53状态外具有相同遗传背景的肿瘤细胞系来研究这些作用对肿瘤细胞p53状态的依赖性。
将转染了突变型TP53（SAS/mp53）或作为对照的neo载体（SAS/neo）的人头颈部鳞状细胞癌细胞皮下接种到Balb/cA裸鼠的两条后腿中。携带肿瘤的裸鼠和携带SCC VII肿瘤的C3H/He小鼠持续接受5-溴-2'-脱氧尿苷（BrdU）以标记肿瘤中的所有P细胞。携带肿瘤的小鼠在未给药、给予尼莫唑、SR-2514或米索硝唑的情况下活着时或肿瘤钳夹后接受γ射线照射，或者在γ射线照射后直接给予未给药、尼莫唑、SR-2514或米索硝唑。对于给药后照射的组，照射后立即切除肿瘤，而对于给药前照射的组，照射后24小时切除肿瘤。将切除的肿瘤切碎并胰蛋白酶消化。将由此获得的肿瘤细胞悬液与细胞松弛素B（一种胞质分裂阻滞剂）一起孵育，并使用针对BrdU的免疫荧光染色来测定未用BrdU标记的细胞（=静止（Q）细胞）中的微核（MN）频率。同时，从未用BrdU预处理的肿瘤中测定总肿瘤细胞群中的MN频率。还在未给予BrdU的小鼠中测定克隆形成细胞存活率。
在这3种肿瘤的总细胞和Q细胞中，有氧和乏氧条件下的放射增敏作用以及γ射线照射后的修复抑制作用均按以下顺序增加：尼莫唑＜SR-2514＜米索硝唑。这两种作用在Q细胞和p53突变肿瘤中分别比在总细胞和p53野生肿瘤中更明显。
在控制放射抗性Q肿瘤细胞和p53突变肿瘤细胞方面，放射增敏剂与传统放疗的联合在放射增敏和修复抑制方面都很有前景，但需要进一步研究对正常组织的毒性。
