Usefulness of combined treatment with mild temperature hyperthermia and/or tirapazamine in the treatment of solid tumors: its independence of 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 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.     

Impact of the p53 status of the tumor cells on the effect of reactor neutron beam irradiation, with emphasis on the response of intratumor quiescent cells.

Human head and neck squamous cell carcinoma cells transfected with mutant p53 (SAS/mp53) or with neo vector as a control (SAS/neo) were inoculated subcutaneously into both the hind legs of Balb/cA nude mice. Tumor-bearing mice received 5-bromo-2'-deoxyuridine (BrdU) continuously to label all proliferating (P) cells in the tumors. After administration of sodium borocaptate-10B (BSH) or p-boronophenylalanine-10B (BPA), the tumors were irradiated with neutron beams. The tumors not treated with 10B-compound were irradiated with neutron beams or gamma-rays. The tumors were then excised, minced and trypsinized. The tumor cell suspensions thus obtained were incubated with a cytokinesis blocker, and the micronucleus (MN) frequency in cells without BrdU labeling (=quiescent (Q) cells) was determined using immunofluorescence staining for BrdU. Meanwhile, 6 h after irradiation, tumor cell suspensions obtained in the same manner were used for determining the frequency of apoptosis in Q cells. The MN and apoptosis frequencies in total (P+Q) tumor cells were determined from the tumors that were not pretreated with BrdU. Without 10B-carriers, in both tumors, the relative biological effectiveness of neutrons was greater in Q cells than in total cells, and larger for low than high cadmium ratio neutrons. With 10B-carriers, the sensitivity was increased for each cell population, especially for total cells. BPA increased both frequencies for total cells more than BSH. Nevertheless, the sensitivity of Q cells treated with BPA was lower than that of BSH-treated Q cells. These sensitization patterns in combination with 10B-carriers were clearer in SAS/neo than in SAS/mp53 tumors. The p53 status of the tumor cells had the potential to affect the response to reactor neutron beam irradiation following 10B-carrier administration.     

Dependency of repair of radiation-induced damage on p53 status and efficient inhibition of the repair with wortmannin treatment in vivo, with reference to the effect on intratumor quiescent cell population

To examine the dependency of p53 status and the effect of wortmannin on the repair of radiation-induced damage, referring to the response of intratumor quiescent (Q) cells. Human head and neck squamous cell carcinoma cells transfected with mutant TP53 (SAS/mp53) or with neo vector (SAS/neo) were injected subcutaneously into left hind legs of nude mice. Tumor-bearing mice received 5-bromo-2'-deoxyuridine (BrdU) continuously to label all intratumor proliferating (P) cells. They received high dose-rate gamma-ray irradiation (HDRI) immediately followed by caffeine or wortmannin administration, or reduced dose-rate gamma-ray irradiation simultaneously with caffeine or wortmannin administration. Nine hours after the start of irradiation, the tumor cells were isolated and incubated with a cytokinesis blocker, and the micronucleus (MN) frequency in cells without BrdU labeling (Q cells) was determined using immunofluorescence staining for BrdU. SAS/neo tumor cells, especially intratumor Q cell populations, showed a marked reduction in sensitivity due to the repair of radiation-induced damage, compared with the total or Q cell populations within SAS/mp53 tumors that showed little repair capacity. In both total and Q cell populations within SAS/neo tumors, wortmannin efficiently suppressed the reduction in sensitivity caused by leaving an interval between HDRI and the assay and decreasing the irradiation dose rate. The repair of radiation-induced damage was thought to be a p53-dependent event. From the viewpoint of tumor control, including intratumor Q-cell control, wortmannin treatment in combination with gamma-ray irradiation is thought to be useful for suppressing the repair of radiation-induced damage.     

Combination of the antivascular agent ZD6126 with hypoxic cytotoxin treatment, with reference to the effect on quiescent tumor cells and the dependency on p53 status of tumor cells

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. Mice bearing the tumors received 5-bromo-2'-deoxyuridine (BrdU) continuously to label all proliferating (P) cells in the tumors. The mice then received a hypoxic cytotoxin, tirapazamine (TPZ) or TX-402, with or without a vascular targeting agent (VTA), ZD6126. Another group of mice given ZD6126 received a series of test doses of gamma-rays while alive or after tumor clamping to obtain hypoxic fractions (HFs) in the tumors. After each treatment, the tumor cells were isolated and incubated with a cytokinesis blocker (cytochalasin-B), and the micronucleus (MN) frequency in cells without BrdU labeling [quiescent (Q) cells] was determined using immunofluorescence staining for BrdU. The MN frequency in total (P+Q) tumor cells was determined from the tumors that were not pretreated with BrdU. Both hypoxic cytotoxins showed significantly greater toxicity toward SAS/mp53 and Q than SAS/neo and total tumor cells, respectively. The sensitivity to TX-402 was significantly higher than that to TPZ in both total and Q tumor cells of both tumors. The significant enhancive effect by ZD6126 combined with each hypoxic cytotoxin was similar irrespective of p53 status, and slightly greater for total than Q cells probably because of a more marked increase in the size of the HFs in total than Q cells on the use of ZD6126 in both tumors, resulting in a reduction of the difference in the sensitivity to the hypoxic cytotoxin between total and Q cells. In the treatment of conventional cancer therapy-resistant Q tumor cells or p53-mutated tumor cells, the use of hypoxic cytotoxin was very promising either alone or when VTA was co-administered. TX-402 might be more promising than TPZ, although further study of the toxicity to normal tissue is needed.     

Reduction of hypoxic cells in solid tumours induced by mild hyperthermia: special reference to differences in changes in the hypoxic fraction between total and quiescent cell populations.

C3H/He mice bearing SCC VII tumours received 5-bromo-2''-deoxyuridine (BrdU) continuously for 5 days via implanted mini-osmotic pumps in order to label all proliferating (P) cells. The tumours were then heated at 40 degrees C for 60 min. At various time points after heating, tumour-bearing mice were irradiated while alive or after being killed. Immediately after irradiation, the tumours were excised, minced and trypsinized. The tumour cell suspensions obtained were incubated with cytochalasin-B (a cytokinesis blocker), and the micronucleus (MN) frequency in cells without BrdU labelling, which could be regarded as quiescent (Q) cells, was determined using immunofluorescence staining for BrdU. The MN frequency in the total (P+Q) tumour cell population was determined from the irradiated tumours that were not pretreated with BrdU. The MN frequency of BrdU unlabelled cells was then used to calculate the surviving fraction of the unlabelled cells from the regression line for the relationship between the MN frequency and the surviving fraction of total (P+Q) tumour cells. In general, Q cells contained a greater hypoxic fraction (HF) than the total tumour cell population. Mild heating decreased the HF of Q cells more markedly than in the total cell population, and the minimum values of HFs of both total and Q cell populations were obtained 6 h after heating. Two days after heating, the HF of total tumour cells returned to almost that of unheated tumours. In contrast, the HF of Q cells did not return to the HF level of unheated tumours until 1 week after heating. It was thought that irradiation within 12 h after mild heating might be a potentially promising therapeutic modality for controlling radioresistant Q tumour cells.     

Impact of the p53 Status of the Tumor Cells on the Effect of Reactor Neutron Beam Irradiation, with Emphasis on the Response of Intratumor Quiescent Cells

Human head and neck squamous cell carcinoma cells transfected with mutant p53 (SAS/mp53) or with neo vector as a control (SAS/neo) were inoculated subcutaneously into both the hind legs of Balb/cA nude mice. Tumor‐bearing mice received 5‐bromo‐2′‐deoxyuridine (BrdU) continuously to label all proliferating (P) cells in the tumors. After administration of sodium borocaptate‐¹⁰B (BSH) or p‐boronophenylalanine‐¹⁰B (BPA), the tumors were irradiated with neutron beams. The tumors not treated with ¹⁰B‐compound were irradiated with neutron beams or γ‐rays. The tumors were then excised, minced and trypsinized. The tumor cell suspensions thus obtained were incubated with a cytokinesis blocker, and the micronucleus (MN) frequency in cells without BrdU labeling (=quiescent (Q) cells) was determined using immunofluorescence staining for BrdU. Meanwhile, 6 h after irradiation, tumor cell suspensions obtained in the same manner were used for determining the frequency of apoptosis in Q cells. The MN and apoptosis frequencies in total (P+Q) tumor cells were determined from the tumors that were not pretreated with BrdU. Without ¹⁰B‐carriers, in both tumors, the relative biological effectiveness of neutrons was greater in Q cells than in total cells, and larger for low than high cadmium ratio neutrons. With ¹⁰B‐carriers, the sensitivity was increased for each cell population, especially for total cells. BPA increased both frequencies for total cells more than BSH. Nevertheless, the sensitivity of Q cells treated with BPA was lower than that of BSH‐treated Q cells. These sensitization patterns in combination with ¹⁰B‐carriers were clearer in SAS/neo than in SAS/mp53 tumors. The p53 status of the tumor cells had the potential to affect the response to reactor neutron beam irradiation following ¹⁰B‐carrier administration.     

Effects of mild temperature hyperthermia and p53 status on the size of hypoxic fractions in solid tumors, with reference to the effect in intratumor quiescent cell populations

PURPOSE: To determine the effects of mild temperature hyperthermia (MTH) and p53 status of tumor cells on the size of hypoxic fractions (HFs) in solid tumors, with reference to the effect on intratumor quiescent (Q) cell populations. METHODS AND MATERIALS: 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 left 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 nicotinamide injection or carbogen gas (95% O(2), 5% CO(2)) inhalation combined with or without MTH. Nicotinamide prevents intermittent blood flow that could induce perfusion-limited acute hypoxia. Chronically hypoxic cells in regions beyond the limitation of oxygen diffusion in tumors are oxygenated by increasing the oxygen transport capacity of circulating blood with carbogen gas inhalation. After each treatment, the mice received a series of test doses of gamma-rays while alive or after tumor clamping to obtain HFs in the tumors. Immediately after irradiation, the tumors were excised, minced, and trypsinized. The tumor cell suspensions thus obtained were incubated with a cytokinesis blocker (cytochalasin-B) to inhibit cytoplasmic division while allowing nuclear division. Tumor cells not labeled with BrdU were detected with immunofluorescence staining of BrdU for P cells, and the micronucleus frequency in cells without BrdU labeling [ = Q cells] was determined. The micronucleus frequency in total (P + Q) tumor cells was determined from the tumors that were not pretreated with BrdU. RESULTS: SAS/mp53 tumors showed larger values for the size of not only the HF but also the diffusion-limited chronically HF than SAS/neo tumors. Q cell populations included a larger HF, particularly the chronically HF, than total cell populations in both tumors, especially in SAS/neo tumors. MTH could efficiently oxygenate the chronically HF, irrespective of p53 status. CONCLUSION: MTH is a useful combined treatment with a radioenhancement effect on intratumor Q cells, irrespective of the p53 status of tumor cells. The p53 status has the potential to affect microenvironmental conditions within solid tumors.     

Radiobiologic significance of apoptosis and micronucleation in quiescent cells within solid tumors following γ-ray irradiation

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

Methods and Materials: 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 γ-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.

Conclusion: γ-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 γ-ray irradiation using P cell labeling with BrdU and the MN frequency assay was also applicable to apoptosis detection assay.     

The usefulness of mild temperature hyperthermia combined with continuous tirapazamine administration under reduced dose-rate irradiation with gamma-rays.

PURPOSE: We clarified the usefulness of mild temperature hyperthermia (MTH) in combination with the continuous administration of tirapazamine (TPZ) under reduced dose-rate irradiation (RDRI) using gamma-rays. MATERIALS AND METHODS: SCC VII tumour-bearing mice received a continuous administration of 5-bromo-2'-deoxyuridine (BrdU) to label all proliferating (P) cells. Then, they received a 24 h continuous subcutaneous infusion of TPZ either with or without MTH under high dose-rate irradiation (HDRI) or RDRI using gamma-rays. After the irradiation, the tumour cells were isolated and incubated with a cytokinesis blocker, and the micronucleus (MN) frequency in non-proliferating tumour cells without BrdU labeling (= quiescent (Q) cells) was determined using immunofluorescence staining for BrdU. The MN frequency in the total tumour cell populations was determined using tumours that were not pretreated with BrdU. RESULTS: The sensitivity of both the total and Q cell populations, especially the latter, was significantly reduced with RDRI compared with HDRI. TPZ increased the sensitivity of both populations, with a slightly more remarkable increase in Q cells. Further, MTH combined with TPZ raised the sensitivity of both the total and Q cell populations, especially the latter, under RDRI more markedly than under HDRI. CONCLUSION: From the viewpoint of solid tumour control as a whole, including intratumour Q-cell control, the use of TPZ, especially in combination with MTH, is useful for suppressing the reduction in the sensitivity of tumour cells caused by the decrease in irradiation dose rate in vivo.     

The usefulness of continuous administration of hypoxic cytotoxin combined with mild temperature hyperthermia, with reference to effects on quiescent tumour cell populations.

To evaluate the usefulness of continuous administration of hypoxic cytotoxins in terms of targeting acute hypoxia in solid tumours and the significance of combination with mild temperature hyperthermia (MTH) (40 degrees C, 60 min), the cytotoxic effects of singly or continuously administered tirapazamine (TPZ) and TX-402 were examined in combination with or without MTH in vivo. Further, the effects were also analysed on total (=proliferating (P)+quiescent (Q)) and Q cell populations in solid tumours with the method for selectively detecting the Q cell response. C3H/He mice bearing SCC VII tumours received a continuous administration of 5-bromo-2'-deoxyuridine (BrdU) for 5 days to label all P cells. The tumour-bearing mice then received a single intra-peritoneal injection or 24 h continuous subcutaneous infusion of hypoxic cytotoxin, TPZ or TX-402, with or without MTH. On the other hand, to detect the changes in the hypoxic fraction (HF) in the tumours by MTH, another group of mice with or without MTH received a series of test doses of gamma-rays while alive or after tumour clamping. After each treatment, the tumour cells were isolated and incubated with a cytokinesis blocker (=cytochalasin-B) and the micronucleus (MN) frequency in cells without BrdU labelling (=Q cells) was determined using immunofluorescence staining for BrdU. The MN frequency in total tumour cells was determined from the tumours that were not pre-treated with BrdU. The sensitivity to TX-402 was slightly higher than that to TPZ in both total and Q tumour cells. Continuous administration elevated the sensitivity of both total and Q cells, especially total cells. MTH raised the sensitivity of Q cells more remarkably than that of total cells in both single and continuous administrations. It was thought to be probably because of the higher dose distribution of hypoxic cytotoxin in intermediately hypoxic areas derived mainly from chronic hypoxia through MTH. From the viewpoint of tumour control as a whole including both total and Q tumour cells, the continuous administration of hypoxic cytotoxin combined with MTH may be useful for sensitizing tumour cells in vivo.     

Heat-induced growth inhibition and apoptosis in transplanted human head and neck squamous cell carcinomas with different status of p53.

To examine p53-dependency in hyperthermic cancer therapy, heat-induced growth inhibition and apoptosis in transplanted human head and neck squamous cell carcinoma (HNSCC) tumours were analysed with different status of p53 into nude mice. The tumour tissue from HNSCC cell line (SAS) transfected with mutant p53 gene (SAS/mp53) or control vector containing neo gene (SAS/neo) was transplanted into the subcutaneous tissue of the thigh of nude mice using a trocar. Hyperthermia was performed at 42 degrees C when the mean diameter of tumour was 5-6mm. The diameter of tumours was measured using vernier calipers and tumour weight (TW) and the relative tumour weight (RW) was calculated. Tumour regrowth delay was evaluated when the RW reached 5-fold against the control group. The accumulation of p53 and Bax proteins was examined by an immunohistochemical technique. Apoptotic cells in the sections were detected by staining of DNA ends using an immunohistochemical technique. SAS/mp53 tumours showed more heat-resistance than SAS/neo tumours. The p53-positively staining cells were observed in untreated SAS/mp53 tumours, but not in untreated SAS/neo tumours. After heat treatment, the accumulation of p53 and Bax proteins was observed in SAS/neo tumours, but little in SAS/mp53 tumours. The incidence of apoptotic cells induced by heat treatment was very low in SAS/mp53 tumours compared with SAS/neo tumours. In conclusion, the heat-induced growth inhibition of a transplanted HNSCC may be correlated with the induction of p53-dependent Bax-mediated apoptosis. Thus, p53 status appears to be one of the useful parameters for the predictive assays in hyperthermic cancer therapy.     

Radiosensitization effect by combination with paclitaxel in vivo, including the effect on intratumor quiescent cells.

PURPOSE: To evaluate the radiosensitization effect on solid tumors upon combination treatment with paclitaxel (TXL), including the effect on intratumor quiescent (Q) cells. METHODS AND MATERIALS: Mice bearing SCC VII or EL4 solid tumors received 5-bromo-2'-deoxyuridine (BrdU) continuously for 5 days to label all proliferating (P) cells. The mice then received gamma-irradiation with or without tirapazamine (TPZ) at various time points after TXL administration. Another group of mice received a series of test doses of gamma-rays while alive or after tumor clamping to obtain hypoxic fractions (HFs) in the tumors at various time points after TXL administration. Immediately after irradiation, the tumor cells were isolated and incubated with a cytokinesis blocker. The micronucleus (MN) frequency in cells without BrdU labeling (Q cells) was determined using immunofluorescence staining for BrdU. Meanwhile, 6 h after irradiation, the tumor cells were isolated from the solid tumors in another group of mice, and 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. For the measurement of the HFs, the MN or apoptosis frequency of Q cells was then used to calculate the surviving fraction of Q cells from the regression line for the relationship between the MN or apoptosis frequency and the surviving fraction of total tumor cells. RESULTS: In both SCC VII and EL4 tumors, maximum values of mitotic index (MI) and apoptosis frequency were observed 9 and 24 h after TXL administration, respectively. However, on the whole, the apoptosis frequency for SCC VII was very low. gamma-Irradiation 9 h after TXL administration induced significant radiosensitization effects on the total cells of both tumors. Irradiation at 60 h had a more significant effect on total cells of EL4 tumor, but no significant effect on total cells of SCC VII tumor. Combined treatment with TXL induced no radiosensitization effect on Q cells in either tumor. The effect on Q cells was observed only after TPZ was administered. The HF of total cells in EL4 tumors decreased significantly 60 h after TXL administration. CONCLUSION: No radiosensitization effect upon combination treatment with TXL is induced in Q tumor cells. However, the effect on P cells is produced by irradiation at the time when the maximum values of MI are induced following TXL administration. In addition, for tumors that are susceptible to apoptosis after TXL administration alone, irradiation at the time of sufficient reoxygenation in tumors after TXL administration produces a greater radioenhancement effect on P cells.     

Evaluation of apoptosis and micronucleation induced by reactor neutron beams with two different cadmium ratios in total and quiescent cell populations within solid tumors.

PURPOSE: Response of quiescent (Q) and total tumor cells in solid tumors to reactor neutron beam irradiation with two different cadmium (Cd) ratios was examined in terms of micronucleus (MN) frequency and apoptosis frequency, using four different tumor cell lines. METHODS AND MATERIALS: C57BL mice bearing EL4 tumors, C3H/He mice bearing SCC VII or FM3A tumors, and Balb/c mice bearing EMT6/KU tumors received 5-bromo-2'-deoxyuridine (BrdU) continuously for 5 days via implanted mini-osmotic pumps to label all proliferating (P) cells. Thirty min after i.p. injection of sodium borocaptate-10B (BSH), or 3 h after oral administration of p-boronophenylalanine-10B (BPA), the tumors were irradiated with neutron beams. The tumors without 10B-compound administration were irradiated with neutron beams or gamma-rays. This neutron beam irradiation was performed using neutrons with two different Cd ratios. The tumors were then excised, minced, and trypsinized. The tumor cell suspensions thus obtained were incubated with cytochalasin-B (a cytokinesis blocker), and the MN frequency in cells without BrdU labeling (=Q cells) was determined using immunofluorescence staining for BrdU. Meanwhile, for apoptosis assay, 6 h after irradiation, tumor cell suspensions obtained in the same manner were fixed, and the apoptosis frequency in Q cells was also determined with immunofluorescence staining for BrdU. The MN and apoptosis frequencies in total (P + Q) tumor cells were determined from the tumors that were not pretreated with BrdU. RESULTS: Without 10B-compounds, the sensitivity difference between total and Q cells was reduced by neutron beam irradiation. Under our particular neutron beam irradiation condition, relative biological effectiveness (RBE) of neutrons was larger in Q cells than in total cells, and the RBE values were larger for low Cd-ratio than high Cd-ratio neutrons. With 10B-compounds, both frequencies were increased for each cell population, especially for total cells. BPA increased both frequencies for total cells more than BSH did. Nevertheless, the sensitivity of Q cells treated with BPA was lower than that of Q cells treated with BSH. Whether based on the MN frequency or the apoptosis frequency, similar results concerning the sensitivity difference between total and Q cells, the values of RBE, and the enhancement effect by the use of 10B-compound were obtained. CONCLUSION: Apoptosis frequency, as well as the MN frequency, can be applied to our method for measuring the Q cell response to reactor neutron beam irradiation within solid tumor in which the ratio of apoptosis to total cell death is relatively high, as in EL4 tumor. The absolute radiation dose required to achieve the same endpoint for Q cells is much higher than that for total cells when combined with 10B-compound, especially with BPA.     

Usefulness of tirapazamine as a combined agent in chemoradiation and thermo-chemoradiation therapy at mild temperatures: reference to the effect on intratumor quiescent cells.

C3H / He mice bearing SCC VII 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 one of six different DNA-damaging agents with or without mild temperature hyperthermia (40 degrees C, 30 min, MTH). These agents were adriamycin (ADM), mitomycin C (MMC), cyclophosphamide (CPA), bleomycin (BLM), cisplatin (CDDP), and tirapazamine (TPZ). After the drug treatment, the tumor-bearing mice were irradiated with a series of doses of gamma-rays. 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 ( = quiescent (Q) cells) was determined using immunofluorescence staining for BrdU. The MN frequency in the total (P + Q) tumor cells was determined from the tumors that had not been pretreated with BrdU. MTH significantly increased the MN frequency of total cells in tumors irradiated with gamma-rays combined with CPA, BLM, CDDP or TPZ, and that of Q cells in tumors irradiated with gamma-rays combined with BLM or TPZ. The sensitivity difference in the MN frequency between total and Q tumor cells was significantly decreased by the combination with TPZ. TPZ combined with radiotherapy and TPZ combined with thermo-radiotherapy at mild temperatures appear to be promising modalities for sensitizing tumor cells in vivo, including Q tumor cells.     

Evaluation of the potential of p-boronophenylalaninol as a boron carrier in boron neutron capture therapy, referring to the effect on intratumor quiescent cells.

C57BL mice bearing EL4 tumors and C3H / He mice bearing SCC VII tumors received 5-bromo-2'-deoxyuridine (BrdU) continuously for 5 days via implanted mini-osmotic pumps to label all proliferating (P) cells. Three hours after oral administration of l-p-boronophenylalanine-(10)B (BPA), or 30 min after intraperitoneal injection of sodium borocaptate-(10)B (BSH) or l-p-boronophenylalaninol (BPA-ol), a newly developed (10)B-containing alpha-amino alcohol, the tumors were irradiated with thermal neutron beams. For the combination with mild temperature hyperthermia (MTH) and / or tirapazamine (TPZ), the tumors were heated at 40 degrees C for 30 min immediately before neutron exposure, and TPZ was intraperitoneally injected 30 min before irradiation. The tumors were then 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 ( = quiescent (Q) cells) was determined using immunofluorescence staining for BrdU. Meanwhile, 6 h after irradiation, tumor cell suspensions obtained in the same manner were used for determining the apoptosis frequency in Q cells. The MN and apoptosis frequency in total (P + Q) tumor cells were determined from tumors that were not pretreated with BrdU. Without TPZ or MTH, BPA-ol increased both frequencies most markedly, especially for total cells. However, as with BPA, the sensitivity difference between total and Q cells was much larger than with BSH. On combined treatment with both MTH and TPZ, this sensitivity difference was markedly reduced, similarly to when BPA was used. MTH increased the (10)B uptake of all (10)B-compounds into both tumor cells. BPA-ol has good potential as a (10)B-carrier in neutron capture therapy, especially when combined with both MTH and TPZ.     

Usefulness of Tirapazamine as a Combined Agent in Chemoradiation and Thermo-chemoradiation Therapy at Mild Temperatures: Reference to the Effect on Intratumor Quiescent Cells

C3H/He mice bearing SCC VII 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 one of six different DNA-damaging agents with or without mild temperature hyperthermia (40°C, 30 min, MTH). These agents were adriamycin (ADM), mitomycin C (MMC), cyclophosphamide (CPA), bleomycin (BLM), cisplatin (CDDP), and tirapazamine (TPZ). After the drug treatment, the tumor-bearing mice were irradiated with a series of doses of γ-rays. 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 (=quiescent (Q) cells) was determined using immunofluorescence staining for BrdU. The MN frequency in the total (P+Q) tumor cells was determined from the tumors that had not been pretreated with BrdU. MTH significantly increased the MN frequency of total cells in tumors irradiated with γ-rays combined with CPA, BLM, CDDP or TPZ, and that of Q cells in tumors irradiated with γ-rays combined with BLM or TPZ. The sensitivity difference in the MN frequency between total and Q tumor cells was significantly decreased by the combination with TPZ. TPZ combined with radiotherapy and TPZ combined with thermo-radiotherapy at mild temperatures appear to be promising modalities for sensitizing tumor cells in vivo, including Q tumor cells.     

Combined effects of tirapazamine and mild hyperthermia on anti-angiogenic agent (TNP-470) treated tumors-reference to the effect on intratumor quiescent cells

PURPOSE: To evaluate the efficacy of the use of tirapazamine (TPZ), especially combined with mild hyperthermia (40 degrees C, 60 min), in the treatment of solid tumors following an anti-angiogenic treatment with TNP-470. In addition, we assessed the effect of TPZ and/or mild hyperthermia (MHT) combined with conventional radiotherapy or chemotherapy on TNP-470 treated tumors. MATERIALS AND METHODS: C3H/He mice bearing SCC VII tumors subcutaneously received TNP-470 at two doses of 100 mg/kg after tumor cell inoculation. At the same time, the tumor-bearing mice 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 TPZ administration combined with or without MHT, gamma-ray irradiation combined with or without TPZ and/or MHT, or cisplatin injection with or without TPZ and/or MHT. Another group of mice received a series of test doses of gamma-rays while alive or after being killed to obtain hypoxic fractions (HFs) in the tumors at various time points after the above-mentioned cytotoxic treatment point. After each treatment, 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 (or quiescent [Q] cells) was determined using immunofluorescence staining for BrdU. The MN frequency in the total (P + Q) tumor cells was determined from the tumors that were not pretreated with BrdU. For the measurement of the HFs, the MN frequency of BrdU-unlabeled cells was then used to calculate the surviving fraction of the unlabeled cells from the regression line for the relationship between the MN frequency and the surviving fraction of total tumor cells. RESULTS: TPZ administration combined with TNP-470 treatment and MHT increased the MN frequency more markedly than treatment with TPZ alone, and this tendency was more remarkable in Q cells than total cells. In both total and Q cells, combined treatment with TPZ and MHT produced significant increases in MN frequencies whether gamma-rays were delivered to TNP-470 treated tumors or cisplatin was injected into the TNP-470 administered mice. Although not significantly, the HFs of total and Q cell populations within solid tumors increased after TNP-470 treatment. CONCLUSION: Combined treatment with TPZ and MHT, whether other cytotoxic treatments such as gamma-ray irradiation or chemotherapy using cisplatin were combined or not, was useful for sensitizing tumor cells in vivo including Q cells even after TNP-470 treatment.     

Reoxygenation in quiescent and total intratumor cells following thermal neutron irradiation with or without (10)B-compound-compared with that after gamma-ray irradiation

PURPOSE: Reoxygenation in quiescent (Q) and total tumor cells within solid tumors after thermal neutron irradiation with or without (10)B-compound was examined, comparing with that following gamma-ray irradiation. METHODS AND MATERIALS: C3H/He mice bearing SCC VII tumors received 5-bromo-2'-deoxyuridine (BrdU) continuously for 5 days via implanted mini-osmotic pumps to label all proliferating (P) cells. Thirty minutes after intraperitoneal injection of sodium borocaptate-(10)B (BSH), or 3 h after oral administration of dl-p-boronophenylalanine-(10)B (BPA), the tumors were irradiated with thermal neutrons, or those without (10)B-compounds were irradiated with thermal neutrons alone or gamma-rays. At various time points after each treatment, a series of test doses of gamma-rays were given to tumor-bearing mice while alive or after being killed to obtain hypoxic fractions in the tumors. Immediately after irradiation, the tumors were excised, minced, and trypsinized. Following incubation of tumor cells with cytokinesis blocker, the micronucleus (MN) frequency in cells without BrdU labeling ( = Q cells) was determined using immunofluorescence staining for BrdU. The MN frequency in the total (P + Q) tumor cells was determined from the tumors that were not pretreated with BrdU. The MN frequency of BrdU-unlabeled cells was then used to calculate the surviving fraction of the unlabeled cells from the regression line for the relationship between the MN frequency and the surviving fraction of total tumor cells. RESULTS: In both total and Q tumor cells, the hypoxic fractions immediately after each treatment went up suddenly. Reoxygenation after each treatment occurred more rapidly in total cells than in Q cells. In both cell populations, reoxygenation appeared to be rapidly induced in the following order: neutron irradiation without (10) gamma-ray irradiation. CONCLUSION: Based on our previous report that total and Q cell fractions within these tumors have larger acutely and chronically hypoxic fractions, respectively, acute hypoxic cells appeared to play a larger role in reoxygenation. BSH was thought to have a potential to distribute more homogeneously in solid tumors than BPA, because BSH induced the nearer reoxygenation pattern to that following neutron irradiation alone than BPA.     

Response of quiescent and total tumor cells in solid tumors to neutrons with various cadmium ratios

Purpose: Response of quiescent (Q) and total tumor cells in solid tumors to neutron irradiation with three different cadmium (Cd) ratios was examined. The role of Q cells in tumor control was also discussed.Methods and Materials: C3H/He mice bearing SCC VII tumors received continuous administration of 5-bromo-2′-deoxyuridine (BrdU) for 5 days using implanted mini-osmotic pumps to label all proliferating (P) cells. Thirty minutes after intraperitoneal injection of sodium borocaptate-¹⁰B (BSH), or 3 h after oral administration of dl-p-boronophenylalanine-¹⁰B (BPA), the tumors were irradiated with neutrons, or those without ¹⁰B-compounds were irradiated with gamma rays. This neutron irradiation was performed using neutrons with three different cadmium (Cd) ratios. The tumors were then excised, minced, and trypsinized. The tumor cell suspensions 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. The MN frequency in total (P + Q) tumor cells was determined from tumors that were not pretreated with BrdU. The sensitivity to neutrons was evaluated in terms of the frequency of induced micronuclei in binuclear tumor cells (MN frequency).Results: Without ¹⁰B-compounds, the MN frequency in Q cells was lower than that in the total cell population. The sensitivity difference between total and Q cells was reduced by neutron irradiation. Relative biological effectiveness (RBE) of neutrons compared with gamma rays was larger in Q cells than in total cells, and the RBE values for low-Cd-ratio neutrons tended to be larger than those for high-Cd-ratio neutrons. With ¹⁰B-compounds, MN frequency for each cell population was increased, especially for total cells. This increase in MN frequency was marked when high-Cd-ratio neutrons were used. BPA increased the MN frequency for total tumor cells more than BSH. Nevertheless, the sensitivity of Q cells treated with BPA was lower than that in BSH-treated Q cells. This tendency was clearly observed in high-Cd-ratio neutrons.Conclusion: From the viewpoint of enhancing the Q-cell sensitivity, tumors should be irradiated with high-Cd-ratio neutrons after BSH administration. However, normal tissue reaction remains to be examined because of its low tumor-to-normal tissue and tumor-to-blood biodistribution ratios.     

Evaluation of the Potential of p-Boronophenylalaninol as a Boron Carrier in Boron Neutron Capture Therapy, Referring to the Effect on Intratumor Quiescent Cells

C57BL mice bearing EL4 tumors and C3H/He mice bearing SCC VII tumors received 5-bromo-2'-deoxyuridine (BrdU) continuously for 5 days via implanted mini-osmotic pumps to label all proliferating (P) cells. Three hours after oral administration of l-p -boronophenylalanine-¹⁰B (BPA), or 30 min after intraperitoneal injection of sodium borocaptate-¹⁰B (BSH) or l-p -boronophenylalaninol (BPA-ol), a newly developed ¹⁰B-containing α-amino alcohol, the tumors were irradiated with thermal neutron beams. For the combination with mild temperature hyperthermia (MTH) and/or tirapazamine (TPZ), the tumors were heated at 40°C for 30 min immediately before neutron exposure, and TPZ was intraperitoneally injected 30 min before irradiation. The tumors were then 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 (=quiescent (Q) cells) was determined using immunofluorescence staining for BrdU. Meanwhile, 6 h after irradiation, tumor cell suspensions obtained in the same manner were used for determining the apoptosis frequency in Q cells. The MN and apoptosis frequency in total (P+Q) tumor cells were determined from tumors that were not pretreated with BrdU. Without TPZ or MTH, BPA-ol increased both frequencies most markedly, especially for total cells. However, as with BPA, the sensitivity difference between total and Q cells was much larger than with BSH. On combined treatment with both MTH and TPZ, this sensitivity difference was markedly reduced, similarly to when BPA was used. MTH increased the ¹⁰B uptake of all ¹⁰B-compounds into both tumor cells. BPA-ol has good potential as a ¹⁰B-carrier in neutron capture therapy, especially when combined with both MTH and TPZ.