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The intrinsic radiation sensitivity of normal and tumour tissue is a major determinant of the outcome of radiotherapy. There is currently no established test that can be used routinely to measure the radiosensitivity of the cells in an individual patient's cancer in a manner that can inform treatment planning. The purpose of this study was to evaluate, in four human colorectal adenocarcinoma cell lines, two possible end points as surrogate markers of radiation response--apoptosis and induction of DNA single-strand breaks--and to compare the results with those of a conventional clonogenic assay. Cell lines (SW707 SW480, SW48 and HT29) known to differ in radiosensitivity were exposed to single doses of X-rays ranging from 0.5 to 5 Gy and cell survival was measured using the clonogenic assay. Apoptosis was determined on the basis of morphology under fluorescent microscopy and DNA damage/repair was measured, as tail moment, using an adaptation of the alkaline comet assay. The relationship between surviving fraction at 2 Gy (SF2) and the percentage of apoptotic cells 24 h after the same dose was complex, but apoptosis accurately predicted the order of radiosensitivities as measured by SF2. Initial damage measured after 2 Gy using the alkaline comet assay gave a close correlation with SF2 (r2=0.95), whereas there was no correlation between initial DNA damage repair rate and SF2.

Bladder tumours show a variable response to radiotherapy with only about 50% showing good local control; currently there is no test to predict outcome prior to treatment. We have used five bladder tumour cell lines (T24, UM-UC-3, TCC-SUP, RT112, HT1376) to investigate the potential of the alkaline comet assay (ACA) to predict radiosensitivity. Radiation-induced DNA damage and repair were compared to clonogenic survival. When the five cell lines were irradiated and initial DNA damage was plotted against cell survival, at all doses (0-6 Gy), a significant correlation was found (r2=0.9514). Following 4 Gy X-irradiation, all cell lines, except T24, showed a correlation between SF2 vs half-time for repair and SF2 vs residual damage at 5, 10, 20 and 30 min. The T24 cell line showed radioresistance at low doses (0-2 Gy) and radiosensitivity at higher doses (4-6 Gy) using both cell survival and ACA end points, explaining the lack of correlation observed for this cell line. These data indicate that initial DNA damage and residual damage can be used to predict for radiosensitivity. Our data suggest that predictive tests of radiosensitivity, appropriate to the clinical situation, may require the use of test doses in the clinical range.

AQ4N is a bioreductive drug that can significantly enhance the anti-tumour effect of radiation and cyclophosphamide. The aim of this study was to examine the ability of AQ4N to potentiate the anti-tumour effect of cisplatin and to compare it to the chemopotentiation effect of tirapazamine. In the T50/80 murine tumour model, AQ4N (50-100 mg/kg) was administered 30 min, 2.5 or 6 h prior to cisplatin (4 mg/kg or 8 mg/kg); this produced an anti-tumour effect that was approximately 1.5 to 2 times greater than that achieved by a single 4 or 8 mg/kg dose of cisplatin. Tirapazamine (25 mg/kg) administered 2.5 h prior to cisplatin (4 mg/kg) resulted in a small increase in anti-tumour efficacy. AQ4N was also successful in enhancing the anti-tumour effect of cisplatin in the SCCVII and RIF-1 murine tumour models. This resulted in an increased cell kill of greater than 3 logs in both models; this was a greater cell kill than that observed for tirapazamine with cisplatin. Combination of cisplatin with AQ4N or tirapazamine resulted in no additional bone marrow toxicity compared to cisplatin administered alone. In conclusion, AQ4N has the potential to improve the clinical efficacy of cisplatin., (Copyright 2001 Cancer Research Campaign.)

The ability of the bioreductive drugs AQ4N and tirapazamine to enhance the anti-tumour effect of cyclophosphamide was assessed in three murine tumour models. In male BDF mice implanted with the T50/80 mammary carcinoma, AQ4N (50-150 mg kg(-1)) in combination with cyclophosphamide (100 mg kg(-1)) produced an effect equivalent to a single 200 mg kg 1 dose of cyclophosphamide. Tirapazamine (25 mg kg(-1)) in combination with cyclophosphamide (100 mg kg(-1)) produced an effect equivalent to a single 150 mg kg(-1) dose of cyclophosphamide. In C3H mice implanted with the SCCVII or RIF-1 tumours, enhancement of tumour cell killing was found with both drugs in combination with cyclophosphamide (50-200 mg kg(-1)); AQ4N (50-200 mg kg(-1)) produced a more effective combination than tirapazamine (12.5-50 mg kg(-1)). Unlike tirapazamine, which showed a significant increase in toxicity to bone marrow cells, the combination of AQ4N (100 mg kg(-1)) 6 h prior to cyclophosphamide (100 mg k(-1)) resulted in no additional toxicity towards bone marrow cells compared to that caused by cyclophosphamide alone. In conclusion, AQ4N gave a superior anti-tumour effect compared to tirapazamine when administered with a single dose of cyclophosphamide (100 mg kg(-1)).

AQ4N (1,4-bis-([2-(dimethylamino-N- oxide)ethyl]amino)5,8-dihydroxyanthracene-9,10-dione) is a novel bioreductive agent that can be reduced to a stable, DNA-affinic compound, AQ4. The alkaline comet assay was used to evaluate DNA damage induced by AQ4N and radiation. Cells prepared from freshly excised T50/80 murine tumours were shown to have the ability to reduce AQ4N to a DNA-damaging agent; this had disappeared within 24 h of excision. When T50/80 tumours implanted in BDF mice were exposed to radiation in vivo a considerable amount of DNA damage was present in tumours excised immediately. Minimal levels of DNA damage were detectable in tumours excised after 2-5 h. AQ4N given 30 min before radiation had no appreciable influence on this effect and AQ4N alone caused only a small amount of damage. When AQ4N and radiation were combined an increasing number of damaged cells were seen in tumours excised 24-96 h after irradiation. This was interpreted as evidence of the continued presence of AQ4, or AQ4-induced damage, which was formed in cells hypoxic at the time of administration of AQ4N. AQ4, a potent topoisomerase II inhibitor, would be capable of damaging cells recruited into the cell cycle following radiation damage to the well-oxygenated cells of the tumour. The kinetics of the expression of the DNA damage is consistent with this hypothesis and shows that AQ4 has persistent activity in vivo.

The haemorheological agent pentoxifylline (PTX) has been shown to improve the relative perfusion and oxygenation of subcutaneous tumours in the mouse. In order to establish whether this effect is dependent on the site of tumour growth, we have looked at changes in the distribution of the cardiac output (COD) to the murine NT carcinoma grown either intradermally (i.d.), intramuscularly (i.m.), on the wall of the caecum, or in all three sites, following i.p. administration of 50 mg kg-1 PTX. In animals bearing a single tumour, PTX treatment significantly increases the COD to tumours located in the caecum, but has no significant effect on the COD to those located in the i.d. or i.m. sites. If all three tumours are present in a single animal, the COD to all three tumours is significantly enhanced by PTX. This appears to reflect the presence of the caecum tumour and does not appear to relate to changes in tumour size or to the haematocrit (HCT) of the blood. We propose that this site dependency implies that a significant increase in blood viscosity only occurs in animals with tumours located in specific sites. Therefore, the potential radiosensitising capability of PTX is highly dependent on tumour location.

Recombinant human erythropoietin (rHuEpo) has recently become available for the treatment of chronic anaemia, including that associated with cancer. Carcinoma NT in CBA mice causes a progressive anaemia which can be overcome by daily injections of recombinant human erythropoietin (rHuEpo). This model was used to study the effect of haematocrit on tumour blood flow, growth rate and radiosensitivity, in mice with haematocrits ranging from approximately 38% (control) to 65% (20 U/day rHuEpo). Tumours showed a small but significant slowing in growth rate with higher haematocrit. In vitro studies showed rHuEpo had no direct effect on the growth of NT cells. Tumour blood flow was measured by two methods in each mouse (133Xe clearance and 86Rubidium uptake). Blood flow showed a tendency to decrease with increasing blood viscosity although this effect was not significant despite the large differences in haematocrit. Although tumour doubling time was prolonged despite the large differences in haematocrit. Although tumour doubling time was prolonged with increasing radiation dose, from 0 (sham irradiated) to 35 Gy, haematocrit was not found to influence the growth delay. This was attributed to adaptation of the tumour during the relatively slow change in the haematocrit. rHuEpo is being considered for clinical use in anaemic cancer patients. Our data suggest that this treatment will correct haematocrit with no effect on tumour radiosensitivity.

The perfusion characteristics of the P22 carcinosarcoma were investigated in tissue-isolated tumour preparations in the ovarian and inguinal fat pads of BD9 rats. Tumours were perfused with a physiological buffer of known viscosity and changes in perfusion pressure were recorded at different perfusion rates in an ex vivo system. At perfusion pressures exceeding 30-40 mmHg tumour flow rate was directly proportional to the perfusion pressure in all tumours, indicating a constant resistance to flow. An apparent positive pressure difference across the tumour vasculature of 20-30 mmHg occurred under conditions of zero flow in either site. At low perfusion pressures, the flow resistance increased sharply due to increases in the geometric resistance of the tumours. These findings are in accord with previously published data. Geometric resistance increased with tumour volume in both sites and was approximately five times greater in the inguinal tumours than it was in the ovarian tumours, on a weight to weight basis. The dependence of tumour geometric resistance on perfusion pressure differs from the situation in normal tissues and may provide a means of manipulating the tumour microcirculation to the exclusion of the systemic blood supply. The dependence of geometric resistance on tumour site may partly explain why tumours located in different sites respond differently to various forms of therapy.

Nicotinamide (NA) and metoclopramide (MCA) have been shown to be sensitisers of the effects of radiation and drugs in experimental rodent tumours growing in skin and muscle. We have used 86Rb uptake to investigate the effects of these two drugs on the distribution of blood to a mouse carcinoma (NT) growing in skin, muscle or the gut wall, as well as to the host normal tissue. NA caused an increase in cardiac output distribution (COD) of between 17 and 92% to tumours in the three sites. When this increase is related to the changes in COD to the host normal tissues, however, COD to tumours in skin and muscle was increased by a factor of 1.8 and to tumours in the gut wall by a factor of 1.7. MCA caused a consistent increase in COD to tumours growing in muscle, but the effects in tumours in skin and gut were variable with time. Again when related to the change in COD to host normal tissues, a factor of 2.1 was seen for COD to tumours growing in muscle and gut. Both NA and MCA alter COD to tumours in some sites relative to host tissues in a way that could enhance anti-cancer drug delivery to tumours, though the effects of NA are more reliable in our systems.

The ability of cancer cells to survive at a distance from blood vessels should be dependent on the local supply of nutrients to each vessel. The corded growth of tumour cells around blood vessels within regions of necrosis in the RH carcinoma in the mouse allows the limit to which cells can be supported by individual vessels to be observed. The thickness of individual tumour cords was measured in conventionally stained tumour sections using a scanning technique to determine the distance between the blood vessel wall and the most distant viable cell adjacent to necrosis. Cord radius was found to vary with the oxygen supply conditions. Control animals had a mean radius of 105 +/- 2 microns while animals that had breathed 10% oxygen had significantly narrower cords (93 +/- 3 microns after 48 h) and animals breathing 100% oxygen had significantly wider cords (117 +/- 3 microns after 24 h). Mice made anaemic (mean hct. 28%) by phlebotomy and plasma transfusion had cord radii that were not significantly different from controls at any time up to 48 h. We conclude that this relatively slow growing mouse tumour is capable of rapid morphological adaptation (less than 3 h) to changes in nutrient availability and that oxygen is probably the limiting substrate.

There is clear clinical evidence that tumours in anaemic patients are difficult to control with radiotherapy. We have studied the radiosensitivity of two transplantable mouse tumours, the SCCVII/St carcinoma and the KHT sarcoma in hosts made anaemic either with an iron poor diet or as a result of tumour growth. The haemoglobin level and haematocrits of mice on the low iron diet fell to about 60% of normal within 11 weeks. The number of clonogenic cells after a single X-ray dose of 20 Gy was slightly lower (P less than 0.05) in the anaemic animals (2.3 X 10(4) g-1) than in controls (5.2 X 10(4) g-1) though there was no significant difference in the surviving fractions. Mice bearing KHT tumours became anaemic with haematocrits falling to 65% of normal as their tumours grew from 300-1200 mg. A second 'test' tumour was implanted one week after the first 'anaemia-inducing' tumour so that estimates of radiosensitivity could all be carried out on tumours within the same size range (150-300 mg). Radiosensitivity was significantly greater in the most anaemic hosts with 2.2 X 10(4) cells g-1 surviving a dose of 20 Gy compared with 6.7 x 10(4) g-1 in controls (P less than 0.01). These results are consistent with most published data for mouse tumours though not for many human tumours.

Enhancement of CCNU cytotoxicity by misonidazole (MISO) was studied in three tumours and two normal tissues in the mouse. The 3 experimental tumours (SCCVII/St, EMT6 and KHT) showed very different sensitivities to CCNU alone, but MISO enhanced the cell killing in ech case. The effect was not always dose-modifying, so that the CCNU dose range for the greatest enhancement was different in each of the tumours. In all 3 tumours, enhancement increased with dose of MISO. The effect on two normal tissues, marrow (CFU-S) and testis (spermatogonia), was also investigated. Enhancement of marrow toxicity could be demonstrated only at CCNU doses greater than 12.5 mg/kg, so that at lower CCNU doses there was a therapeutic gain equal to the tumour enhancement ratio. The spermatogonia effect, however, showed enhancement by MISO similar to that seen in the tumours at all CCNU doses up to 20 mg/kg.

The response of 14 mouse tumour sub-lines to the radiosensitizing action of a large single dose of misonidazole (MISO) has been assessed by regrowth delay. In 13 of these, significant enhancement of radiation effect occurred under ambient conditions, indicating sensitization of naturally hypoxic cells. The enhancement observed (SER') varied with the radiation dose, as would be predicted for a mixed oxic/hypoxic cell population. The maximum SER' in these 13 tumours did not depend on histology or regrowth rate. The 14th tumour, a slow-growing sarcoma, was not sensitized under ambient conditions, but showed marked sensitization when clamped to produce acutely hypoxic cells. This is consistent with no hypoxic cells occurring naturally in a sarcoma with a slow rate of growth. Faster-growing variants of this tumour showed radiosensitization under ambient conditions. The slow-growing carcinoma, RH, however, appears to contain hypoxic cells and did show sensitization. The cytotoxic action of MISO was compared with the radiosensitization by administering it after irradiation in 8 of the tumour lines. In 2 tumours no cytotoxicity was observed. In the rest cytotoxicity was significant, but much smaller than the sensitization observed when MISO was administered before irradiation. These regrowth-delay data have been used to calculate hypoxic fractions in 3 ways. Estimates of hypoxic fraction ranged from less than 0.1% in the slow sarcoma to greater than or equal to 30% in several tumours. There is considerable variation in the estimate, according to the technique used.

Experiments were carried out to determine whether the enhancement of alkylating-agent cytotoxicity seen after large single doses of misonidazole (MISO) in mouse tumours can also be achieved by prolonged exposure to low MISO levels similar to those which can be tolerated clinically. The level in mouse blood plasma could be maintained at about 100 micrograms/ml for 7 h by injecting small doses of MISO every 1/2 h. The effect of this treatment in combination with cyclophosphamide (CY) or melphalan (L-PAM) was studied in the RIF-1 tumour, using regrowth delay and cell-survival cloning assays. In each case, prolonged exposure to low levels of MISO gave enhancement ratios very close to those obtained with a large single dose. ERs of 1.6-2.0 were obtained with CY and 1.8-2.2 with L-PAM over the range of alkylating-agent doses used. In experiments with CY the response of 2 normal-tissue systems, marrow and WBC count, was also studied. No significant enhancement of CY damage occurred in either case. In the L-PAM experiments the LD50/30 and WBC counts were determined as normal-tissue end points. Multiple MISO had no effect. Our results show that levels of MISO which can be achieved safely in man yield good enhancement of the tumour cytotoxicity of 2 widely used chemotherapeutic agents without increasing the damage to normal tissues.

The ability of the calcium antagonists, cinnarizine and flunarizine, to enhance the radiosensitisation produced by the administration of an erythrocyte transfusion to anaemic, RIF-1 or SCCVII/St tumour bearing mice was determined. Erythrocyte transfusion alone increased radiation cell killing 10-fold in the RIF-1 tumour when given 0-4 h before X-rays. In contrast, the SCCVII/St showed only a 4-fold increase in sensitivity, apparent when erythrocytes were given 2-6 h before irradiation. The administration of 50 mg kg-1 cinnarizine or flunarizine to anaemic mice followed by erythrocyte transfusion 0 h before X-rays produced the same level of cell survival for both tumours, a 20-fold increase in cell killing for cinnarizine, and a 30-40-fold effect for flunarizine, even though at this time interval, the erythrocyte transfusion alone did not sensitise the SCCVII/St tumour to X-rays. Further investigations indicated, however, that the erythrocyte transfusion was necessary to achieve the sensitisation with the calcium antagonists, since giving flunarizine to anaemic mice alone only achieved a 4-fold increase in radiation cell killing. In addition, flunarizine given with erythrocyte transfusion 4 h before X-rays, in SCCVII/St, the optimal time for radiosensitisation in this tumour, did not further increase the level of cell killing achieved by flunarizine plus erythrocyte transfusion 0 h before X-rays.

The effect of misonidazole (MISO) on the cytotoxicity of cyclophosphamide (CY) was investigated in the mouse. The response of the RIF-1 tumour was measured by growth delay and by cell survival in a cloning assay. MISO enhanced the cytotoxicity of CY. For single treatment, enhancement was maximal when MISO was given 30 min to 2 h before CY. The enhancement ratio (i.e. the dose of CY alone divided by the dose of CY with MISO required to cause the same response) increased with increasing dose of MISO up to 250 mg/kg, but decreased with increasing dose of CY above 50 mg/kg. For 5 daily treatments, enhancement increased with CY dose up to approximately 25 mg/kg/injection. Survival of marrow stem cells was measured using the spleen-colony assay. MISO did not enhance significantly the cytotoxicity of CY at doses under 100 mg/kg. Enhancement was seen at higher doses, but the effect was less than in tumours. CY reduced the number of circulating white blood cells. Neutrophils were most severely depleted. The WBC count was slightly lower when CY was given in combination with MISO than after CY alone, but the effect could be accounted for by direct MISO cytotoxicity. These experiences suggest that a therapeutic gain may be achieved if MISO is combined with doses of CY in the clinical range. From experiments performed to investigated the possible mechanisms involved, we conclude that for the RIF-1 tumour the major effect of MISO is to inhibit the repair from CY-induced potentially lethal damage.

The effect of the calcium antagonists, cinnarizine and flunarizine on the radiation sensitivity of two murine tumours, RIF-1 and SCCVII/St was investigated. Initial experiments giving the compounds at 50 mg kg-1 i.p. indicated that cinnarizine had no effect on cell survival after 20 Gy of X-rays in the RIF-1 sarcoma and only a small effect in the SCCVII/St carcinoma. However, flunarizine produced a small radiosensitisation in the RIF-1 tumour and a substantial sensitisation in the SCCVII/St tumour. Subsequent experiments in the SCCVII/St tumour indicated that the optimal radiosensitising dose of flunarizine was approximately 5 mg kg-1, although some sensitisation was apparent throughout the range of 0.05-500 mg kg-1. Flunarizine produced a parallel shift in the X-ray dose response curve, equivalent to a 5-fold reduction in hypoxic fraction. In a normal tissue study, 5 mg kg-1 flunarizine did not enhance the reduction in white cell counts produced by X-ray doses of 2-8 Gy. These data suggest that flunarizine may have some potential use as a radiosensitiser.

The effect of inhibitors of nuclear ADP-ribosyl transferase (ADPRT) on the cytotoxicity of melphalan (L-PAM) in the RIF-1 tumour in vivo was investigated. A large single dose of nicotinamide (1000 mg kg-1) enhanced the tumour cell killing by L-PAM as measured by tumour cell survival. This enhancement was maximum when nicotinamide was administered within 1 h before injecting the L-PAM. When given at this time, the nicotinamide had a dose-modifying effect on all L-PAM doses tested, giving rise to a mean enhancement ratio (ER) of 2.2. Nicotinamide did not appear to inhibit the recovery from L-PAM induced potentially lethal damage. L-PAM (6 mg kg-1) produced a transient drop in mouse body temperature. This effect was both increased and prolonged by nicotinamide. In addition the inhibitor also delayed the clearance of L-PAM from the plasma of C3H mice, such that the half-life of the chemotherapeutic agent was extended from 41 min to 143 min. The effect of combining L-PAM with nicotinamide doses below 1000 mg kg-1 was also investigated. The results showed that as the nicotinamide dose was decreased, the enhancement of the effects on body temperature, pharmacokinetics and white blood cell counts were reduced. However, a concomitant loss in the enhancement of tumour cell killing was also observed. Similar results were obtained using 3-aminobenzamide, a more efficient inhibitor of ADPRT.

The ability of a range of 2-nitroimidazoles with similar electron affinities but widely differing partition coefficients (P) to enhance the cytotoxicity of cyclophosphamide (CY) in mouse tumour and normal tissues was investigated. In a preliminary study large single doses of benznidazole (BENZ), misonidazole (MISO), desmethylmisonidazole (DMM), and SR-2508 were found to give similar enhancement of the RIF-1 and SCC VII/St tumours. SR-2555 was less effective. A direct comparison was made between MISO and SR-2508 using prolonged, low-level drug exposures, achieved by multiple injections. The enhancement of CY cytotoxicity achieved in the two tumour systems (RIF-1 and SCC VII/St) was found to be similar for a given blood sensitizer concentration. In the normal tissue assays (white blood cell count, bone marrow CFU-S and testis spermatogonia) neither MISO nor SR-2508 produced significant enhancement of CY cytotoxicity, so that the therapeutic gains achieved at a given blood concentration of sensitizer were similar for SR-2508 and MISO. The main advantage of SR-2508, however, will probably lie in its lower toxicity, permitting higher blood levels to be achieved. However, the slope of the dose response curves are rather shallow so we would not predict a dramatically increased benefit.

Appropriate control of the affinity of haemoglobin for oxygen is fundamental to the efficient oxygenation of our tissues. Important modifiers of this relationship are pH, CO2 concentration and the intraerythrocytic level of 2,3-diphosphoglycerate (2,3-DPG). We have studied the influence of haemoglobin affinity on the radiosensitivity of the RIF-1 sarcoma in the mouse. Changes in haemoglobin affinity were induced by exposing donor mice to either 10% oxygen, normal air, or 100% oxygen for 48 h. Blood was drawn from these animals and exchanged transfused into tumour-bearing mice immediately before irradiation. Transfusion of blood from mice breathing 10% oxygen carried a lowered haemoglobin affinity and produced marked radiosensitization of the tumours in the recipients; transfusion with normal blood had no significant effect and transfusions from mice breathing 100% oxygen caused a small increase in radioresistance. Measurements of the level of 2,3-DPG in the blood of these groups showed higher concentrations in the oxygen-deprived animals than in controls but no significant change in animals exposed to 100% oxygen. These results demonstrate that alterations in haemoglobin affinity, probably resulting from changes in 2,3-DPG levels, can have a powerful influence on tumour radiosensitivity. We feel that this mechanism could have considerable clinical importance.

The effect of the nitroimidazoles misonidazole, Ro-05-9963, RGW-608 and metronidazole on nerve conduction velocity (NCV) were measured in the anaesthetized mouse. The compounds were administered by i.p. injection either as a single dose of 1 mg/g (only 0.5 mg/g for RGW-608) or in 36 fractions of 0.15 mg/g over 18 days (only 4 fractions in 2 days for RGW-608). After single doses a reduction in nerve conduction velocity was seen with all the compounds except metronidazole, which had no significant effect. During chronic exposure, a reduction in NCV occurred towards the end of the course of injections. All compounds produced an effect, although RGW-608 was the most neurotoxic, giving the largest reduction in NCV after only 4 injections. After the end of chronic exposure to misonidazole, Ro-05-9963 and metronidazole, recovery to normal took 2-3 weeks.