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The effect of combinations of the conventional chemotherapeutic agent 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) and nitroimidazole radiation sensitizers was evaluated in female C3H mice. Tumour response to single-agent or combination therapy was assessed in a tumour growth-delay assay. In the KHT sarcoma the simultaneous addition of misonidazole (MISO) was found to increase significantly the tumour growth delay resulting from CCNU treatment. The observed enhancement ratios (ER) increased with MISO dose, and ranged from 1.3 to 1.9 for sensitizer doses of 0.25-1.0 mg/g. The combination of CCNU and 1.0 or 0.5 mg/g MISO in the RIF-1 tumour or the MT-1 tumour produced ERs of approximately 2.0 and approximately 1.5 respectively. In the KHT sarcoma a series of other nitroimidazole sensitizers, including Ro-05-9963, SR-2555, SR-2508 and metronidazole (METRO), were also evaluated at equimolar doses (5 mmol/kg) in combination with a 20mg/kg dose of CCNU. Unlike MISO, these compounds in general failed to enhance the CCNU cytotoxicity in this tumour model. However, SR-2508 did enhance the response of the RIF-1 tumour to large single doses of CCNU, though not as much as MISO. Normal-tissue toxicity was determined using peripheral white blood cell (WBC) counts 3 days after treatment. CCNU doses of 10-50 mg/kg given either alone or in simultaneous combination with 0.5 or 1.0 mg/g MISO were studied. WBC toxicity increased with CCNU dose, but the addition of MISO at either dose did not significantly enhance this normal-tissue toxicity.

We have studied in detail the relationship between glutathione (GSH) depletion and sensitivity of HEp3 human carcinoma cells to doxorubicin [Adriamycin (ADR)]. Exponentially growing HEp3 cells were incubated with L-buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, for different periods so that a range of GSH depletion could be obtained. These GSH-depleted cells were then treated with a combination of BSO and ADR (1 microgram/ml) for various durations. Under these conditions, the cytotoxicity of ADR was significantly enhanced by GSH depletion. The extent of ADR dose enhancement was found to be inversely proportional to cellular GSH level at the time of ADR treatment. Furthermore, it was shown that the dose-enhancement factors (DEF) also correlated with the duration of combined BSO and ADR treatment. For example, at a GSH level of 45% of untreated control, 18.5 +/- 3 fmol/cell or 4.8 +/- 0.3 X 10(-3) fmol/mum3 (+/- SD), DEF of 8.0, 6.4, and 5.0 were obtained for treatment periods of 3 hours, 2 hours, and 1 hour, respectively. Further study showed that the GSH kinetics differed significantly for the different treatment times, which indicates that GSH kinetics may be an important factor in determining the intrinsic sensitivity of HEp3 cells to ADR. Furthermore, the kinetics of GSH response to ADR varied significantly between cell lines. In the study of the effect of such differences, the GSH kinetics of 3 human ovarian tumor cell lines with different intrinsic sensitivities to ADR were investigated.(ABSTRACT TRUNCATED AT 250 WORDS)

We have used monochlorobimane as a quantitative marker by which cells of naturally high or low GSH contents were purified by fluorescence-activated cell sorting (FACS). The cell line chosen for this purpose, MLS, was a human ovarian tumor cell line established from a patient who had received extensive chemotherapy and showed evidence of 'multidrug' resistance. Cells of a specified volume were sorted into subpopulations containing the 1% most dim (low GSH) and 1% most bright (high GSH) cells. With an increasing number of sortings, cell subpopulations emerged with progressively lower (dim) and higher (bright) GSH content as compared to the parent population. After 4 sortings, GSH contents were 10.6 +/- 0.8, 5.1 +/- 0.4, and 7.2 +/- 0.7 X 10(-18) moles/micron3 for MLS/bright, MLS/dim and MLS/parent respectively. The high and low GSH phenotypes were of limited stability reverting to the parent phenotype by the sixth week following the last FACS. Cells with high GSH content were more resistant to adriamycin than cells with low GSH content, for example, at 1 log cell kill MLS/bright was 1.6 fold more resistant than MLS/dim. An ADR resistant variant of the MLS line, designated MLS/ADRR/2, established by twice treating MLS cells with 1 microgram/ml ADR for 2 hr, also showed increased GSH content (1.3-fold) and ADR resistance as compared with the parent line. These results illustrate the possible importance of tumor cell GSH status in determining the response to chemotherapy of a heterogenous population of tumor cells with diverse GSH contents.

The KHT sarcoma is a model system in which metastases can be studied in multiple organs without prior clonal selection. The present series of experiments were designed to evaluate and compare the extent of potentiation of chemotherapeutic agent activity by radiosensitizers when KHT sarcoma cells were grown in different anatomical sites. In these studies the effect of combining misonidazole (MISO) and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) on KHT lung nodules and ovarian metastases was determined. Ovarian metastases were a consequence of the direct spread of tumor cells growing as lung nodules. Once established, KHT cells in the lungs and ovaries grew with a similar doubling time (1-2 days). Response of tumors at either site to chemotherapy in the presence or absence of a sensitizer was assessed using an in vivo to in vitro excision assay. MISO (1.0 mmol/kg) was administered simultaneously with a range of CCNU doses and survival of clonogenic tumor cells was measured 24 h after treatment. The results demonstrate that the addition of MISO to CCNU treatment potentiated the action of this chemotherapeutic agent at both tumor sites although greater cell kill enhancement occurred in the ovarian metastases. In the lung nodules, when combined with CCNU, a 1.0 mmol/kg dose of MISO was found to yield a dose modifying factor (DMF) of approximately 1.3. The same combination resulted in a DMF of approximately 1.8 in the ovarian metastases. This difference in DMF was not a result of an intrinsic difference in sensitivity to CCNU since cells grown at either site gave rise to the same dose response curve. Rather the difference in dose modification by MISO appears to be a consequence of the larger fraction of radiobiologically hypoxic cells in the ovarian tumors (approximately 50 per cent) than in the lung nodules (approximately 5 per cent) at the time of treatment. These findings suggest the use of drug-sensitizer combinations to treat disseminated disease and provide further evidence for the requirement of hypoxia in chemopotentiation by radiosensitizers.

Patients with ovarian cancer often respond well to combination chemotherapy initially but the majority eventually relapse when, with further treatment, the initially successful regimen proves ineffectual. The cause of such failures frequently has been attributed to the development of drug resistance. Although the mechanisms of acquired resistance in situ are still poorly understood, studies in vitro have shown that cells selected for resistance to one drug often exhibit cross-resistance to other seemingly unrelated agents, suggesting a somewhat generalised mechanism of resistance. We have studied the role of glutathione (GSH) and drug transport in determining the sensitivity to adriamycin (ADR) of a panel of human ovarian cell lines established directly from biopsies of patients with diverse treatment histories. These cell lines exhibited inherent differences in sensitivity to ADR by a dose factor of up to 3; a difference that was considerably less than what has been reported when cells were selected for drug resistance in vitro. The differences in drug sensitivity reported here among the various cell lines appeared to be unrelated to drug transport, in terms of both influx and efflux. Moreover, although these cell lines have a wide range of GSH content, there was only a poor correlation between drug sensitivity and cellular GSH content per se. However, when exposed to a clinically relevant dose of ADR, the GSH content of cell lines that were 'sensitive' decreased, whereas that of cell lines that were 'resistant' increased. To take these time-dependent changes in GSH into consideration, the area under the GSH content versus time curve (AUC), with and without ADR treatment, was calculated for each cell line. When this latter factor was included in the analysis, greatly improved correlations were found between GSH kinetic parameters and responses to ADR. In particular, ADR resistance was found to be closely correlated with the positive changes in absolute GSH AUC following ADR treatment (r = 0.92; P less than 0.01). Using 35S-labelled cysteine and methionine as tracers, it was found that the essential difference between the 'resistant' and 'sensitive' lines was that the 'resistant' lines had higher steady-state rates of GSH synthesis than the 'sensitive' lines. These results demonstrate that changes in cellular GSH concentration during treatment may be an important indicator of tumour cell response to ADR.

The degree of non-neoplastic host-cell infiltration was assessed in 3 in vivo-in vitro tumour models commonly used in radiobiological studies: EMT6/Ro mammary carcinoma, 9L/Ro tumour and KHT sarcoma. While the 2 former tumour models have been shown to be moderately to highly immunogenic when grown s.c., the KHT sarcoma is apparently non-immunogenic. Using differential staining on single-cell suspensions from enzymatically dissociated solid tumours, all 3 tumour types were found to contain large proportions (30-60%) of non-neoplastic host cells. The actual host-cell component found in the cell suspensions differed both in type and percentage for the 3 tumours studied. These host and neoplastic cells in the cell suspensions prepared from the solid tumours could be readily separated by centrifugal elutriation. After separation the clonogenic potential of the neoplastic cells was assessed, and was found to be higher than the clonogenic capacity of the unseparated cell suspension by a factor directly related to the host/neoplastic cell ratio. Even after the removal of the host cells, the clonogenic capacities of the neoplastic EMT6 and 9L tumour cells were lower than that of the corresponding in vitro sublines (approximately 30 vs 75%). However, in the KHT sarcoma the removal of the host cell component raised the plating efficiency to approximately 60%, which was similar to the value for the in vitro cell subline of this tumour.

Tumor response to radiation is dependent not only on the quantity of hemoglobin (Hb) available for oxygen (O2) transport but also on the position of the Hb-O2 dissociation curve (Hb affinity). Previous studies have shown that administering agents which shift the Hb-O2 dissociation curve to the right (decrease Hb affinity) sensitize tumors to radiation by reducing the fraction of radiobiologically hypoxic cells. However, there may be toxicity limitations when agents aimed at altering Hb affinity are administered directly to the host. The present studies evaluated the therapeutic benefit of shifting the Hb-O2 dissociation curve in vitro prior to the transfusion of the biochemically modified RBCs into recipient hosts. Mice were given a hemolysis agent (phenylhydrazine hydrochloride, PH) prior to transfusing RBCs with normal or altered Hb affinity. A 100 mg/kg dose of PH reduced the hematocrit to approximately 60% of control 24 hr after treatment. Tumors irradiated at this time demonstrated an increased fraction of hypoxic cells. If the hematocrit was returned to normal by transfusing mice prior to irradiation, a significant but transient reduction in the hypoxic fraction was seen. Tumor response was reduced if RBCs with elevated Hb affinity, obtained by storing the erythrocytes at 4 degrees C, were used. Alternatively, tumor sensitization was noted when animals were transfused with RBCs having decreased Hb affinities. The latter was achieved by incubating the RBCs in the presence of either clofibrate or the precursors of 2,3 diphosphoglycerate (2,3 DPG). These findings further support the notion that the Hb affinity is an important parameter in determining tumor response to radiation and suggest that this factor ought to be considered when RBCs are used to transfuse anemic patients undergoing radiotherapy.

Studies were performed to determine whether the radiation sensitizer misonidazole (MISO) could enhance the tumor control probability in a treatment strategy combining radiation and the nitrosourea 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU). In initial experiments KHT sarcoma-bearing mice were injected with 1.0 mg/g of MISO simultaneously with a 20 mg/kg dose of CCNU 30-40 min prior to irradiation (1500 rad). These timings were chosen to maximize the effectiveness of MISO both as a chemopotentiator and radiosensitizer. With this treatment protocol approximately 60% of the mice were found to be tumor-free 100 days post treatment. By comparison all 2 agent combinations led to 0% cures. To evaluate the relative importance of chemopotentiation versus radiosensitization in the 3 agent protocol, tumors were treated with MISO plus one anti-tumor agent (either radiation of CCNU) and then at times ranging from 0 to 24 hr later exposed to the other agent. When the time between treatments was 0 to 6 hr, a 60 to 80% tumor control rate was achieved for both MISO plus radiation followed by CCNU and MISO plus CCNU followed by radiation. However if the time interval was increased to 18 or 24 hr, the cure rate in the former treatment regimen dropped to 10% while that of the latter remained high at 40%. These results were not due to the radiation-CCNU sequence but rather reflected the ability of the sensitizer to act as a chemopotentiator when CCNU is given 0 to 6 hr after the MISO-radiation combination. This was not the case when the MISO-radiation combination was administered 18 or 24 hr prior to CCNU. The data therefore indicate that 1) improved tumor responses may be achieved when MISO is added to a radiation-chemotherapy combination and 2) MISO may be more effective in such a protocol when utilized as a chemopotentiator.

Surfactant precursors or other products of Type II pneumocytes have the potential to be the first biochemical marker for late radiation effects. This is particularly clinically important in the combined modality era because of the frequent occurrence of pneumonitis and pulmonary fibrosis secondary to radiation or chemotherapy. Accordingly, correlative studies have been pursued with the Type II pneumocyte as a beginning point to understand the complex pathophysiology of radiation pneumonitis and fibrosis. From our ultrastructural and biochemical studies, it is evident that Type II pneumocytes are an early target of radiation and the release of surfactant into the alveolus shortly after exposure persists for days and weeks. Through the use of lavaging techniques, alveolar surfactant has been elevated after pulmonary irradiation. In three murine strains and in the rabbit, there is a strong correlation with surfactant release at 7 and/or 28 days in vivo with later lethality in months. In vitro studies using cultures of type II pneumocytes also demonstrate dose response and tolerance factors that are comparable to the in vivo small and large animal diagnostic models. New markers are being developed to serve as a predictive index for later lethal pneumonopathies. With the development of these techniques, the search for early biochemical markers in man have been undertaken. Through the use of biochemical, histological, and ultrastructural techniques, a causal relationship between radiation effects on type II pneumocytes, pulmonary cells, endothelial cells of blood vessels, and their roles in the production of pneumonitis and fibrosis will evolve.

Isoeffect analysis of complete dose response curves can be used to study the interaction of agents in combined modality protocols. When such an analysis is applied to data from in vivo tumour model systems, the effects of the agents on factors such as tumour vasculature, growth or reoxygenation pattern also need to be considered. In this study the change in tumour size, which can occur during a long time-interval between agents, was used as an example of a factor which may influence the position of data points on an isoeffect plot. Assays of in vivo tumour growth delay and in vitro clonogenic survival were performed to demonstrate that the radiation response curves of EMT-6/Ro tumours were size dependent. These curves were used to illustrate that data points obtained from a combined modality treatment may fall outside of the envelope of additivity of an isoeffect plot, as a direct consequence of tumour growth. This finding indicates that it may not be possible to interpret the results from isoeffect analyses of in vivo data on the basis of cellular interactions between agents, and suggests that instead isoeffect analyses be applied primarily to assess the overall response of the tumour system.

The potential therapeutic advantage of including the radiosensitizer misonidazole (MISO) in a treatment regimen combining systemic chemotherapy and localized radiotherapy was assessed in the KHT sarcoma. Tumor-bearing C3H mice were treated with 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, administered either alone or in combination with MISO, 24 h prior to irradiating the tumors with doses ranging from 0-35 Gy. The sensitizer exposure was either a single dose of 2.5 mmol/kg or multiple injections given every 0.5 h for 8 h to maintain a sensitizer blood level of approximately 100 micrograms/ml. The chemotherapeutic agent was administered simultaneously with the single sensitizer dose or 3 h into the chronic sensitizer dosing schedule. Tumor responses to the combined modality treatments was determined using end points of tumor regrowth delay and animal tumor-free survival. Skin reactions in the treatment field were scored as a measure of normal tissue complications. MISO, administered as a single dose in the combined modality therapy, enhanced the tumor response by a factor of approximately 1.8-1.9 while increasing skin reactions approximately 1.1- to 1.2-fold. Combining 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea with chronic MISO administration prior to irradiation increased the resultant tumor control probability 1.5- to 1.8-fold without enhancing normal tissue complications. Consequently using MISO as a chemopotentiator in a 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea-radiation combination can yield a significant therapeutic benefit.

Previous investigations have shown that combining the radiation sensitizer misonidazole with conventional alkylating chemotherapeutic agents can lead to a therapeutic advantage. More recently, another sensitizer, RSU 1069, has been reported to give an enhancement of antitumor agent efficacy similar to that observed with misonidazole, but at an approximately tenfold lower sensitizer dose. One chemotherapeutic agent whose activity has been modified by sensitizers to a greater extent in tumors than in critical normal tissues is the nitrosourea lomustine (CCNU). The present studies evaluated the therapeutic benefit of combining RSU 1069 and CCNU in KHT sarcoma-bearing C3H/HeJ mice. The drugs were administered ip, and tumor response was assessed by measuring the survival of clonogenic KHT cells 22-24 hours after treatment. Normal tissue toxicity was determined using peripheral wbc counts 3 days after treatment and a 30-day lethality assay. Combining CCNU with a 0.38-mmol/kg dose of RSU 1069 increased tumor cell killing by a factor of approximately 1.9. Wbc toxicity and 30-day animal lethality increased with CCNU dose, but the addition of RSU 1069 enhanced either endpoint only slightly (factor of 1.0-1.2). The addition of RSU 1069 to CCNU treatment, therefore, led to a significant therapeutic benefit.

The immediate release of surfactant into lung alveoli following irradiation has been studied as a potential indicator for the later development of radiation pneumonitis. Utilizing single dose radiation exposure to the whole thorax in male LAF1/J mice, steep dose response curves for lavaged alveolar surfactant were identified at 7 and 28 days after exposure. Seven days after irradiation there was no elevation with doses up to and including 12 Gy; above this dose a detectable increase occurred. At 28 days the surfactant recovered by lavage was elevated compared to the levels seen at day 7 for all doses; doses greater than 12 Gy produced surfactant values significantly greater than those found in mice treated with 12 Gy or less. The radiation pulmonary lethality dose response curve assessed four months later indicated an LD50 value of approximately 13 Gy. The early biochemical effect and the later radiation pneumonitis lethalities therefore closely coincided. The evidence strongly indicates that alveolar surfactant release uncovered hours to days after radiation exposure may be an early biochemical marker that predicts for subsequent pneumonitis radiation injury.

Direct comparison of the cell age response of 9L and KHT tumour cells derived either from tissue culture or solid tumours was achieved. Cells from dissociated KHT and 9L tumours (the latter implanted either subcutaneously or intracerebrally) and cells from tissue culture were separated into homogeneous sized populations by centrifugal elutriation. In both tumour models these homogeneous sized populations correspond to populations enriched at different stages of the cell cycle. The survival of these elutriated cell populations was measured after a single dose of Cs-137 gamma rays. For cells isolated from 9L solid tumours, there was little variation in radiosensitivity throughout the cell cycle; however, a very small but significant increase in resistance was found in late G1 cells. This lack of a large variation in radiosensitivity through the cell cycle for 9L cells from solid tumours also was seen in 9L cells growing in monolayer tissue culture. When similar experiments were performed using the KHT sarcoma tumour model, the results showed that KHT cells in vitro exhibited a fairly conventional increase in radioresistance in both mid G1 and late S. However, the cell age response of KHT cells from solid tumours was different; particularly in the late S and G2 + M phases. These data demonstrate that direct extrapolation of in vitro cell age responses to the in situ situation may not always be valid.

Cellular glutathione (GSH) levels were compared in human and rodent tumor cells grown both in vivo and in vitro. Three human (A431, HEp3, ME180) and two murine (KHT and RIF-1) tumor cell lines were used. The average GSH contents for exponentially growing human cells in vitro were 14.2, 10.9, and 17.0 fmol/cell for ME180, A431, and HEp3 cells, respectively. These cells also were grown as tumors in nude mice. Following dissociation, greater than 90% pure populations of neoplastic and nonneoplastic cells were isolated by centrifugal elutriation prior to GSH determination. The data showed that the GSH levels of the tumor associated host cells were appreciably lower than those of the neoplastic cells. In addition, in contrast to the values obtained for the exponential cells, neoplastic cells grown in vivo showed a 2- to 3-fold reduction in GSH. However, the values for in vivo cells were similar to those obtained for the same tumor cells grown in vitro in the plateau phase. Compared to the human tumor cells the GSH contents of murine tumor cells always were lower. For example, RIF-1 and KHT cells in the exponential growth phase had GSH contents of 3.3 and 7.5 fmol/cell, respectively. Also, as was observed with the human cells, the GSH content of KHT cells in plateau phase of growth was 2-3 times less than that of cells in the exponential phase of growth. Similarly, the GSH content of KHT cells grown as in situ tumors prior to dissociation and isolation by centrifugal elutriation also was reduced by a factor of 3 compared to exponential phase cells. Although the average volume of tumor cells grown in vivo was less than that of cells grown in vitro, this did not account for the differences in GSH values observed when in vitro and in vivo derived cells were compared. Finally, GSH measurements made on multiple biopsies of individual human tumor xenografts varied by a factor of 2-3 within each tumor type studied. This variation, likely due to host cell fluctuations, may present a complicating feature in the interpretation of solid tumor GSH levels.

A previous study by the same authors has reported the effect of acute reductions in the arterial partial pressure of oxygen (PaO2) on tumour response to radiation. The results have been extended in the present paper to investigate tumour response to radiation in animals in which the PaO2 is chronically reduced. The purpose of these experiments was to simulate the condition of cancer patients undergoing radiotherapy in the presence of chronically low PaO2 values as might be expected in patients with chronic respiratory disease. Mice bearing transplantable KHT sarcomas were kept in a 12% O2 environment prior to (10--16 days), during and following the radiation treatment of their tumours. During the period of low PaO2 (about 50 mm Hg) exposure, the mice were found to increase their haemoglobin (Hg) levels by approximately 50%. Because of this increase, the response, determined using a growth delay assay of the tumours irradiated at reduced PaO2 was found to be the same as that observed for tumours in mice breathing air throughout the experiment. In mice with reduced PaO2 levels maintained at normal Hb concentrations by periodic bleeding, tumour response was found to be similar to that of mice with acute PaO2 reductions. These results indicate that chronic PaO2 reductions in the absence of Hb compensation may have a detrimental effect on the success of a radiation treatment.

The effect of including a nitroimidazole in a treatment regimen combining two alkylating chemotherapeutic agents was evaluated in a mouse tumor model. KHT sarcoma-bearing female C3H/HeJ mice were treated with a single dose of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) either alone or in combination with the radiosensitizer misonidazole (MISO) prior to a range of cyclophosphamide (CY) doses. CCNU (5 mg/kg) preceded CY treatment by 3 hr. MISO (1 mmol/kg) was given 1 hr after CCNU (2 hr prior to CY). Tumor response was assessed using either an in vitro to in vivo clonogenic cell survival assay 22-24 hr after treatment or an in situ delay of tumor regrowth assay. The results demonstrated that the inclusion of MISO at a dose of 1.0 mmol/kg increased tumor cell kill resulting from the combination of CCNU-CY by a factor of 1.4-1.5 compared to that seen in the absence of the nitroimidazole. Normal tissue toxicity resulting from the CCNU-CY or CCNU-MISO-CY combinations was determined by measuring bone marrow stem cell (CFU-S or CFU-GM) toxicity. Compared to CCNU-CY alone, the addition of MISO did not enhance this normal tissue toxicity. These findings indicate that the inclusion of MISO in a combination chemotherapy protocol may yield a significant therapeutic benefit.

The effectiveness of localized X-irradiation on the transplantable KHT sarcoma was investigated while the host mice breathed gas mixtures ranging from 5% O2 : 95% N2 to 100% O2. Subcutaneous tumours in the flank of C3H mice anaesthetized with Nembutal (sodium pentobarbitol), Valium (diazepam), or urethane, were irradiated with doses of 1,500 or 2,500 rads and the delay in days for the tumours to grow from 4 to 10 mm in diameter was determined. Measurements of the arterial partial pressure of oxygen (PaO2) were made under conditions identical to the irradiation experiments. For PaO2 values less than 100 mm Hg (animals breathing air), growth delay decreased linearly, while for PaO2 values greater than 100 mm Hg, growth delay increased and reached a plateau at a PaO2 between 200 and 300 mm Hg, (animals breathing congruent to 50% O2). This relationship holds for the two doses and the three anaesthetics studied, and indicates the importance of investigating the Pa02 levels of patients undergoing radiotherapy treatment.

We have investigated in detail the effects of buthionine sulfoximine (BSO), a selective glutathione (GSH) depleting agent, on the GSH contents of a number of normal tissues and three experimental tumours in mice. Significant variations in the rate and degree of GSH depletion and recovery were observed among the normal tissues. Following a dose of 2.5 mmol kg-1 BSO, GSH nadirs were reached by approximately 5 h for the liver and kidney, 8 h for the lung and bone marrow, 12 h for red blood cells (RBCs) and by 24 h for the heart. The degree of depletion was greatest for the kidney (80%), liver (74%) and bone marrow (83%), intermediate for the heart (54%) and lung (40%), and least for RBCs (13%). Recovery of GSH content was fastest for the liver followed in descending order by the kidney, the lung, the bone marrow, RBCs and the heart. In contrast, the rate and extent of GSH depletion and recovery showed considerably less variation among the 3 murine tumours. In the tumours GSH nadirs were reached by 10-12 h. The extent of depletion was about 55-65%. Recovery of GSH levels in the tumours required 48 h or more, a longer period than required by the liver, kidney and lung but shorter than that needed for the bone marrow, heart and RBCs. Attempts to preferentially deplete tumour GSH by exploiting the differences in recovery rates between normal tissues and tumours were only partially successful. Multiple BSO dosing at 16 h intervals allowed the liver to recover between doses, but the recovery in the kidney, lung and bone marrow was only partial and no recovery was seen in the heart. Finally, dose-depletion relationship investigations showed that, with the exception of the lungs, GSH depletion could be achieved in tumours with doses of BSO lower than those required for normal tissues.

By centrifugal elutriation, viable cell subpopulations were isolated directly from KHT sarcomas treated with 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, mitomycin (MIT C), cyclophosphamide (CTX), or radiation. Survival in these subpopulations was determined by a clonogenic cell survival assay either immediately (radiation) or 24 hours after treatment with drugs. Following irradiation, late S and G2/M cells were found to be sensitive, but G1 cells were preferentially spared. For all chemotherapeutic agents evaluated, survival of cells at the G1/S boundary 24 hours after drug exposure was at a nadir, whereas cells in the late S and G2/M at this time were always drug resistant. The response of cells in early G1 24 hours after drug treatment ranged from sensitive (MIT C) to resistant (CTX). Because of these results, the combination of MIT C and radiation was investigated in detail. When MIT C was administered 24 hours before tumor irradiation, the chemotherapeutic agent significantly reduced the radiation-resistant subpopulations, whereas the radiation treatment effectively killed cells spared by the chemotherapeutic agent. Isobologram analysis further indicated that this treatment combination led to "supra-additive" cell kill in the tumor.

The effect of combining the radiosensitizer misonidazole (MISO) with 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) prior to radiation on the response of various KHT sarcoma cell subpopulations was evaluated. Centrifugal elutriation was used to obtain homogeneous populations of cells with respect to their cell cycle position from suspensions prepared directly from treated solid KHT tumors. Survival in these subpopulations was determined using an in vivo to in vitro cloning assay. In situ treatment consisted of either (a) CCNU (3.0 mg/kg) plus MISO (1.0 mg/g), (b) radiation (15 Gy) alone, or (c) CCNU plus MISO preceding the radiation by 24 hr. In the latter protocol, one treatment effectively eliminated those cells being preferentially spared by the other. The CCNU-MISO followed by radiation combination not only reduced the treatment resistance due to hypoxia and cell cycle position but also left all cell subpopulations in the tumor equally sensitive to the treatment. To determine the nature of the interaction between these agents in detail, the extent of cell killing following such a treatment regimen was evaluated using isoeffect plot (isobologram) analysis. KHT sarcoma-bearing C3H mice were treated with various doses of CCNU either alone or in simultaneous combination with a 1.0-mg/g dose of MISO 24 hr prior to receiving a range of radiation doses. Combining CCNU and radiation led to significantly enhanced tumor cell killing. When evaluated by isoeffect plot analysis, the data points resulting from this combination fell well below the envelope of additivity, indicating that a supraadditive interaction between these two agents had occurred in the tumor cells. An even greater interaction was observed in the KHT sarcomas when MISO was combined with CCNU prior to irradiation. The findings of effective elimination of treatment-resistant subpopulations along with a supraadditive tumor cell kill offer, at least in part, an explanation for the previously described therapeutic advantage observed for the CCNU-MISO-radiation protocol.

The effects of fractionated doses, in vivo thiol modulation, and antifibrinolytic therapy on the expression of lung damage induced by cyclophosphamide (Cy) were evaluated in C3H mice. The protein content of lung lavage samples taken 4 days after Cy treatment was used as an early indicator of damage. In fractionation studies, little difference in lung protein was observed when 200 mg of Cy/kg was administered as a single dose or as two or four equal doses given daily, suggesting that little sparing effect occurred with fractionated doses of Cy. In experiments that tested the effects of exogenous thiol administration, mice treated with WR-2721 before Cy were protected against lung damage, whereas the use of sodium thiosulfate or mesna did not give this protection. Treatment with epsilon-aminocaproic acid, which inhibits the breakdown of fibrin clots, did not result in enhanced Cy damage as measured by lung lavage or breathing rate; this suggests that the extended presence of fibrin per se did not contribute to Cy-induced pulmonary damage.

The potentiation of chemotherapeutic agents by radiation sensitizers has been extensively studied for several years. There is little doubt that the effectiveness of certain anti-cancer drugs, primarily alkylating agents, can readily be enhanced both in vitro and in vivo through the addition of a sensitizer. While enhanced effects have been observed in certain critical normal tissues, in general most animal model studies have demonstrated a therapeutic gain at large sensitizer doses. This approach to combination therapies therefore appears promising. Yet many questions concerning the interaction between chemotherapeutic agents and radiosensitizers, particularly in the area of mechanisms of action, still remain. This overview attempts to focus on some of these questions. Four aspects of modification of chemotherapy by nitroimidazoles are reviewed and discussed. These address the importance in chemopotentiation of i) hypoxia, ii) alterations in DNA damage and/or repair, iii) depletion of intracellular sulfhydryls and iv) modification of drug pharmacokinetics. It is concluded that: i) even though chemopotentiation can occur at intermediate oxygen levels, hypoxia ultimately plays a pivotal role, ii) no single unifying mechanism for chemopotentiation exists; alterations in drug pharmacokinetics, cellular SH levels and DNA damage/repair all are involved, the relative importance of each factor is dependent on the particular drug-sensitizer combination, iii) it is important to continue the evaluation of chemopotentiation under conditions mimicking clinically achievable sensitizer pharmacokinetics and iv) further investigations into more effective utilization of chemopotentiation are warranted.

Previous investigations have shown that the combination of the conventional chemotherapeutic agent lomustine (CCNU) and the nitroimidazole radiation sensitizer misonidazole can lead to an improved therapeutic result. To study whether altered CCNU metabolism plays a role in this chemopotentiation, mice were treated with known modifiers of hepatic microsomal enzymes prior to CCNU exposure, and tumor response and systemic toxicity were assessed. KHT sarcoma-bearing C3H/HeJ mice were pretreated with either (a) five daily doses of phenobarbital (80 mg/kg) to induce the microsomal enzymes and then CCNU 48 hours later or (b) a 50-mg/kg dose of the microsomal enzyme inhibitor SKF 525A 1 hour before CCNU. Tumor response and normal tissue toxicity following treatment were measured using a regrowth delay and 30-day lethality assay, respectively. Phenobarbital pretreatment reduced both the antitumor efficacy and toxicity of CCNU (factor of approximately 0.8), while SKF 525A increased the effect of CCNU in both cases (factor of approximately 1.6). Thus, unlike pretreatments with the sensitizer misonidazole, pretreatments with phenobarbital or SKF 525A did not result in a therapeutic advantage for CCNU.

Studies were performed to determine the location, with respect to cell cycle phase, of the radiobiologically hypoxic cells in KHT sarcomas. Cells dispersed from solid KHT tumours were separated into subpopulations at different stages of the cell cycle by centrifugal elutriation. Flow cytometric analysis of the DNA content of these subpopulations indicated that the degree of synchrony that could be achieved was greater than or equal to 95% for G1 cells, 70-75% for S cells and 70-75% for G2M cells. The approach to locate and characterize hypoxic cells was based on the premise that due to their lack of oxygen such cells would preferentially survive radiation treatment. Consequently KHT sarcomas were irradiated in situ either in dead animals or in animals breathing air. Following treatment, the tumours were dissociated, the cells elutriated into the various phases of the cell cycle and clonogenic cell survival was determined. Complete dose-response curves were determined for cells in the G1, S and G2M cell cycle phases. The various cell cycle subpopulations obtained from tumours irradiated while mice breathed air, all demonstrated survival curves with a break and final slope which paralleled that of the corresponding anoxic cell survival curve. From these curves the proportion of hypoxic tumour cells in the G1 phase was calculated to be 10.1 +/- 1.7%. Because the elutriated S and G2M enriched cell fractions were to some extent contaminated by cells from other phases of the cell cycle, the percentage of hypoxic S and G2M tumour cells was estimated to range from 0-7% and 0-5% respectively. However, since G1 cells comprised the majority of all the neoplastic cells in these tumours, the data suggest that hypoxic cells in KHT sarcomas are found primarily in this cell cycle stage.