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 hind legs of Balb/cA nude mice. Mice bearing the
tumors received
5-bromo-2'-deoxyuridine (
BrdU) continuously to label all proliferating (P) cells in the
tumors. The mice then received
tirapazamine (TPZ) with or without mild temperature
hyperthermia (40 degrees C, 60 min) (MTH), gamma-ray irradiation with or without MTH and/or TPZ,
cisplatin (CDDP) with or without MTH and/or TPZ, or
paclitaxel (TXL) with or without MTH and/or TPZ. After each treatment, the
tumors were excised, minced and trypsinized. The
tumor cell
suspensions thus obtained were incubated with a cytokinesis blocker (
cytochalasin-B), and the micronucleus (MN) frequency in cells without
BrdU labeling (i.e., quiescent (Q) cells) was determined by using immunofluorescence staining for
BrdU. Meanwhile, 6 h after gamma-ray irradiation or 24 h after other cytotoxic treatments,
tumor cell
suspensions obtained in the same manner were used for determining the frequency of apoptosis in Q cells. The MN frequency and apoptosis frequency in total (P+Q)
tumor cells were determined from the
tumors that were not pretreated with
BrdU. On the whole, gamma-ray irradiation and CDDP injection induced a higher frequency of apoptosis and lower frequency of MN in SAS/neo cells than SAS/mp53 cells. There were no apparent differences in the induced frequency of apoptosis and MN between SAS/neo and SAS/mp53 cells after TPZ or TXL treatment. MTH sensitized cells to TPZ-inducing cytotoxicity more markedly in SAS/mp53 and Q cells than in SAS/neo cells and total cells, respectively. In gamma-ray irradiation and CDDP treatment, the enhancement in combination with MTH and/or TPZ was more remarkable in SAS/mp53 cells and Q cells than in SAS/neo and total
tumor cells, respectively. Also in the case of TXL treatment, the combination with MTH and/or TPZ induced a slightly greater enhancement effect in SAS/mp53 cells and Q cells. In view of the difficulty in controlling mutated p53 status
tumors and intratumor Q cells, combination treatment with MTH and/or TPZ as a cooperative modality in
cancer therapy is considered to have potential for controlling solid
tumors as a whole.