The cell transmembrane pH gradient in tumors enhances cytotoxicity of specific weak acid chemotherapeutics.

The extracellular pH is lower in tumor than in normal tissue, whereas their intracellular pH is similar. In this study, we show that the tumor-specific pH gradient may be exploited for the treatment of cancer by weak acid chemotherapeutics. i.v.-injected glucose substantially decreased the electrode estimated extracellular pH in a xenografted human tumor while its intracellular pH, evaluated by (31)P magnetic resonance spectroscopy, remained virtually unchanged. The resulting increase in the average cell pH gradient caused a parallel increase in tumor growth delay by the weak acid chlorambucil (CHL). Regardless of glucose administration, the effect of CHL was significantly greater in tumors preirradiated with a large dose of ionizing radiation. This suggests that CHL was especially pronounced in radioresistant hypoxic cells possessing a larger transmembrane pH gradient. These results indicate that the naturally occurring cell pH gradient difference between tumor and normal tissue is a major and exploitable determinant of the uptake of weak acids in the complex tumor microenvironment. The use of such drugs may be especially effective in combination with radiation.

The general relation between tissue response to x-radiation (alpha/beta-values) and the relative biological effectiveness (RBE) of protons: prediction by the Katz track-structure model.

PURPOSE: Experimental data suggest that the relative biological effectiveness (RBE) of protons compared to x-rays may be determined by the alpha/beta-ratio of the x-ray survival curve. The data are referring to the centre of a spread-out Bragg peak (SOBP) formed to deliver a homogeneous dose to the tumour by modulating the proton energy. In an effort to explore the basis for this observation, calculations were performed to investigate the response of different biological targets through a range of proton energies and doses. MATERIALS AND METHODS: To describe the x-ray survival curve, the parameters of the linear-quadratic equation, alpha and beta, as well as those of the multi-target/single-hit equation, n and D0, were considered. These parameters were varied to investigate the RBE using the Katz track-structure model. Known cell line characterizations, as well as different hypothetical cells assuming different alpha/beta-ratios but similar target size parameters in the framework of the track-structure theory, were considered. RESULTS: The RBE was found to increase with increasing alpha/beta when the parameter n was varied, but to decrease with increasing alpha/beta when D0 was varied. This held when all other radiosensitivity parameters were assumed to stay constant. Thus, the RBE cannot be predicted by the alpha/beta-ratio alone. CONCLUSIONS: Although there is no direct correlation between the proton RBE and the parameters describing the x-ray survival curve, the track-structure model predicts a tendency for late-responding tissues (low alpha/beta) to have higher RBE values than early-responding tissues (high alpha/beta). These calculations reinforce the experimental findings, but also strongly suggest that there are circumstances in which the tendency for RBE to increase with increasing alpha/beta does not occur, or even could be reversed.

Relative biological effectiveness of proton beams in clinical therapy.

PURPOSE: In clinical proton beam radiation therapy, an RBE of 1.1 relative to megavoltage X-rays is currently being employed at most treatment centers. This RBE pertains to radiation in the spread out Bragg-peak (SOBP) for all tissue systems, all dose levels per fraction and all proton beam energies. As the number of centers and treatment sites for which proton beam therapy continues to increase and additional experimental data is accrued, a re-assessment of the justification for a generic RBE is warranted. In this paper we address: (1) the constancy of the RBE along the central axis from the plateau entrance to the distal SOBP (upstream of the distal edge); (2) RBE as a function of dose (or cell survival level); and (3) the target cell or tissue (alpha/beta) dependency of the RBE. This analysis pertains to modulated proton beams of initial energies of approximately 70-200 MeV and SOBPs of approximately 2-10 cm, respectively. RESULTS AND CONCLUSIONS: With exceptions, the available experimental data indicate that the RBE of SOBP protons increases with decreasing dose or dose per fraction and increasing depth in the SOBP, with the magnitude of both effects likely being dependent on the alpha/beta ratios of the target cells or tissues. The use of a generic RBE of 1. for all tissues, especially those exhibiting low alpha/beta values such as CNS, may be too low, especially at dose levels of < or = 2 Gy/fraction. Systematic determination of the RBE values dependent upon the three interdependent variables identified in this manuscript (beam depth, dose size and target tissue) will provide an enhanced data base for detailed treatment planning and institutional trial comparisons, thereby maximizing the therapeutic benefit of proton beams.

The pH partition theory predicts the accumulation and toxicity of doxorubicin in normal and low-pH-adapted cells.

The accumulation and toxicity of the weak base doxorubicin has been investigated as a function of extracellular pH, intracellular pH and the cellular pH gradient in cells previously cultured under normal (pH 7.4) and low-pH (6.8) conditions. Low-pH-adapted cells exhibit transmembrane pH gradients which substantially differ from normal cells at the same extracellular pH. No relationship was obtained between intracellular pH and the uptake or toxicity of doxorubicin in the two cell types. In contrast, doxorubicin accumulation and toxicity increased with increasing extracellular pH in both normal and low-pH-adapted cells. However, at the same extracellular pH, drug cytotoxicity was more pronounced in normal than in low-pH-adapted cells. The difference in doxorubicin accumulation and cytotoxicity at the same extracellular pH was found to be dependent on the difference in the transmembrane pH gradient of the two cell types. As the cellular pH gradient differs between tumour and normal tissue, this observation suggests a basis for enhancing cellular drug uptake in either tissue type.

Enhancement of fractionated-dose irradiation by retinoic acid plus interferon.

PURPOSE: To evaluate the relative cytotoxicity of fractionated-dose radiation in the presence and absence of 13-cis-retinoic acid (RA) plus alpha-2a-interferon (IFN), as a function of overall treatment time. METHODS AND MATERIALS: Studies were performed with the human squamous cell carcinoma line FaDu, in vitro. Attached exponential phase cells were treated with RA + IFN for 8-10 h and then exposed to single graded doses of radiation, or 1 to 6 doses of radiation at 2 Gy per dose, or to 5 doses of radiation at 2 Gy/dose with a time interval of 4-24 h between treatments. Following irradiation, the cells were incubated with drugs present throughout colony formation, and the fraction of survivors in the presence and absence of the combined drugs was calculated. RESULTS: For single graded-dose irradiation, the surviving fraction ratio at 2 Gy in the absence vs. presence of drugs was 1.27 +/- 0.19 in 3 repeat experiments. Following administration of 6 doses of radiation at 2 Gy/fraction with a 5-h time interval between treatments and, after correcting for cell proliferation between treatments, the surviving fractions differed by a factor of 3.25, again indicating an average difference in survival of 1.26 after each of the 6 2-Gy/fractions. Treatment with 5 2-Gy doses of irradiation with 24 vs. 4 h elapsing between doses, resulted in a 3-fold greater decrease in survival in the presence of drugs vs. no drug. The relatively greater cell kill due to 24 vs. 4 h between treatments was due to drug inhibition of cell proliferation between the more prolonged treatments. CONCLUSIONS: The results of this study indicate that retinoic acid plus interferon both sensitizes and inhibits cell proliferation during treatment. These results suggest that this combination of radiation and drugs, when used concurrently, may be effective for inhibiting tumor cell proliferation or accelerated repopulation during clinical fractionated radiotherapy.

Cytotoxicity of weak electrolytes after the adaptation of cells to low pH: role of the transmembrane pH gradient.

Theory suggests that the transmembrane pH gradient may be a major determinant of the distribution of lipophilic weak electrolytes across the cell membrane. The present study evaluates the extent to which this factor contributes to pH-dependent changes in the cytotoxicity of two such chemotherapeutic drugs: chlorambucil and mitoxantrone. Experiments were performed with two cell types of the same origin but exhibiting different pH gradients at the same extracellular pH (pHe): CHO cells cultured under normal physiological conditions (pH 7.4) and acid-adapted cells obtained by culturing under low pH conditions (6.8). Over the pHe range examined (6.0-7.6), the difference between intracellular pH (pHi) and pHe increased with decreasing pHe. Acid-adapted cells were more resistant to acute changes in pHi than normal cells, resulting in substantially larger gradients in these cells. Drug cell survival curves were performed at pHe values of 6.4, 6.8 and 7.4. The cytotoxicity of chlorambucil, a weak acid, increased with decreasing pHe, and low pH-adapted cells were more sensitive than normal cells at the same pHe. In contrast, for the weak base, mitoxantrone, cytotoxicity increased with pHe and was more pronounced in normal cells. As predicted by the theory, the cytotoxicity of both drugs changed exponentially as a function of the pH gradient, regardless of cell type. For mitoxantrone, the rate of such change in cytotoxicity with the gradient was approximately two times greater than for chlorambucil. This difference is probably due to the presence of two equally ionizable crucial groups on mitoxantrone vs one group on chlorambucil. It is concluded that the cellular pH gradient plays a major role in the pH-dependent modulation of cytotoxicity in these weak electrolytes. The data obtained also suggest that a pronounced differential cytotoxicity may be expected in vivo in tumour vs normal tissue. In comparison with normal cells at a pHe of 7.4 (a model of cells in normal tissues), acid-adapted cells at a pHe of 6.8 (a model of cells distal from supplying blood vessels in tumours) were more sensitive to chlorambucil, with a dose-modifying factor of approximately 6, and were more resistant to mitoxantrone by a factor of 14.

Tumor pH: implications for treatment and novel drug design.

Although limited data exist, electrode-measured pH values of human tumors and adjacent normal tissues, which are concurrently obtained by the same investigator in the same patient, consistently show that the electrode pH (believed to represent tissue extracellular pH primarily) is substantially and consistently lower in tumor than in normal tissue. In contrast, the 31P-magnetic resonance spectroscopy-estimated intracellular pH is essentially identical or slightly more basic in tumor compared with normal tissue. As a consequence, the cellular pH gradient is substantially reduced or reversed in these tissues. This difference provides an exploitable avenue for the treatment of cancer. The extent to which drugs exhibiting weakly acid or basic properties are ionized depends on their ionization potential (pKa) and the pH of their milieu. Weakly acidic drugs that are lipid soluble in their nonionized state diffuse freely across the cell membrane and on entering a relatively basic intracellular compartment become trapped and accumulate within the cell. This may lead to substantial (10-fold or more) differences in the intracellular-to-extracellular drug distribution between tumor and normal tissue for cytotoxics, hypoxic cell sensitizers, or other drugs exhibiting appropriate pKa. Experimental in vitro evaluation of these predictions confirms both the predicted pH gradient-dependent changes in cellular drug accumulation and toxicity.

The effect of androgen deprivation and radiation therapy on an androgen-sensitive murine tumor: an in vitro and in vivo study.

PURPOSE: The purpose of this research was to assess whether or not tumor eradication by irradiation can be enhanced by prior androgen deprivation in an androgen-dependent rodent tumor model. MATERIALS AND METHODS: The androgen-sensitive Shionogi SC-115 mouse mammary carcinoma was grown in athymic male NCr/Sed (nu/nu) mice and in culture. An androgen-deprived environment was created in vivo by surgical orchiectomy and in vitro through the use of charcoal-stripped androgen-free medium. The dose of radiation required to control 50% of tumors (tumor control dose [TCD50] assay) was used to assess the radiation response of tumors grown in vivo. Colony-formation assays were used for in vitro assessment. RESULTS: After orchiectomy, Shionogi tumors regress in volume quickly but inevitably regrow as androgen-insensitive clones. The TCD50 for 6-mm Shionogi tumors grown in intact mice was 89.0 Gy (95% confidence intervals [CI]: 83.4 to 94.9). When orchiectomy was performed 24 to 48 hours prior to irradiation, the TCD50 fell to 60.3 Gy (95% CI: 54.8 to 66.3). When irradiation was withheld until maximum tumor regression following orchiectomy, it was lower still-42.1 Gy (95% CI: 37.4 to 47.4). In vitro studies demonstrated no profound changes in intrinsic radiation sensitivity induced by prior androgen deprivation, although there was a trend toward lower Dzero values with increasing duration of deprivation. DISCUSSION: Prior androgen deprivation enhances the ability of irradiation to eradicate Shionogi tumors in vivo. It is likely that this effect results from cytoreduction and/or improvement in the nutritional status of a smaller tumor, but changes in intrinsic radiation sensitivity cannot be excluded.

Effect of basic fibroblast growth factor on angiogenesis and growth of isografted bone: quantitative in vitro-in vivo analysis in mice.

Basic fibroblast growth factor (bFGF), a constituent of bone and cartilage matrix, has been shown to be a potent mitogen for osteoblasts and chondrocytes and yet an inhibitor of chondrocyte terminal differentiation in cell culture. To characterize the effect of bFGF on bone formation, whole neonatal murine femora were cultured in the presence or absence of bFGF and a neutralizing antibody against bFGF. In vitro, femoral elongation was provided by cartilage growth only; the calcified diaphyseal zone stained by oxytetracycline did not increase. When bFGF was added to the culture medium, longitudinal growth of the proximal and distal cartilage was inhibited in a dose-dependent manner (p < 0.05), and the number of hypertrophic chondrocytes in the growth plate was reduced. This phenomenon was absent in the presence of a neutralizing antibody, which when given alone significantly promoted femoral elongation. In contrast, in vivo after transplantation into adult mice bearing dorsal skin fold chambers, femora rapidly calcified after revascularization. This observation supports the notion that bone formation largely depends on angiogenesis-mediated events. To verify this hypothesis, angiogenesis and bone formation were quantified using bFGF known to be a stimulator of angiogenesis. Calcification of grafted femora was accelerated by bFGF given intraperitoneally. The neutralizing antibody slightly suppressed angiogenesis and femoral elongation (not statistically significant), whereas intravenous injections of both substances did not reveal a significant modulatory effect. In vivo the effect of systemically administered bFGF was inhomogeneous, but there was a strong correlation between angiogenesis and endochondral calcification (p < 0.001). These results suggest that exogenous bFGF modulates bone formation in vitro by inhibition of terminal differentiation of chondrocytes in the growth plate, and angiogenesis and concomitant in vivo events are pivotal in the promotion of rapid bone formation.

Heating or freezing bone. Effects on angiogenesis induction and growth potential in mice.

We have characterized the effect of bone graft treatment by heating or freezing (with or without dimethyl sulfoxide (DMSO)). Tissue culture and dorsal skin-fold chambers in mice were used as sites to quantify the effect on angiogenesis, growth and calcification of neonatal femora. Fresh femora increased in both length and cartilage diameter (calcification in vivo only), but cryopreservation or heating abolished the increase in femoral dimensions. In vivo, femora of all experimental groups elicited an angiogenic response from the host tissue, which was most pronounced for fresh femora, weaker for DMSO-preserved frozen bone and poor for unprotected frozen bone and boiled femora. Freezing in the presence of a cryopreservative (DMSO) was found to preserve the angiogenic potential of frozen bone, whereas unprotected heating or freezing significantly impaired angiogenesis induction and growth potential.

The reproducibility and relationship between single and fractionated dose estimates of the surviving fraction at 2 Gy.

PURPOSE: To evaluate the reproducibility and relationship between the surviving fraction at 2 Gy obtained from single-graded dose and multifraction survival curve assays. METHODS AND MATERIALS: Single-graded dose and multifraction survival curve assays were concurrently performed in five human cell lines. For the multifraction studies, five to six doses at 2 Gy per fraction were administered with a time interval of 5.5 h between fractions. All surviving fraction data was corrected for multiplicity and cell proliferation during treatment. Replicate experiments were performed for each cell line. RESULTS: The precision of the Sf2 estimates obtained from multifraction studies was approximately three times greater than was obtained in the single-dose studies. For the single-dose studies, the average difference between the Sf2s obtained in the initial vs. repeat experiment was 0.11; for the fractionated-dose studies the difference was significantly reduced to 0.032. A rank-order correlation was not obtained between the 2 Gy Sfs in the initial and repeat single-dose assays; in the multifraction studies, the correlation was significant (p < 0.05). The rank-order correlation between the single- and fractionated-dose Sf2 assays was significant only when the two sets of assays were pooled; however, the coefficient of correlation remained low (R(2) = 0.50). CONCLUSIONS: The precision of the estimates of the surviving fraction at 2 Gy, obtained from multifraction assays, was substantially greater than was obtained in single dose-survival curve assays.

Cellular pH gradient in tumor versus normal tissue: potential exploitation for the treatment of cancer.

Although limited data exist, electrode-measured pH values of human tumors and adjacent normal tissues, which are concurrently obtained by the same investigator in the same patient, consistently show that the electrode pH (believed to primarily represent tissue extracellular pH) is substantially and consistently lower in the tumor than in normal tissue. In contrast, the 31P-magnetic resonance spectroscopy estimated that intracellular pH is essentially identical or slightly more basic in tumor compared to normal tissue. As a consequence, the cellular pH gradient is substantially reduced or reversed in tumor compared to normal tissue: in normal tissue the extracellular pH is relatively basic, and in tumor tissue the magnitude of the pH gradient is reduced or reversed. The difference provides an exploitable avenue for the treatment of cancer. The extent to which drugs exhibiting weakly acid or basic properties are ionized is strongly dependent on the pH of their milieu. Weakly acidic drugs which are relatively lipid soluble in their nonionized state may diffuse freely across the cell membrane and, upon entering a relatively basic intracellular compartment, become trapped and accumulate within a cell, leading to substantial differences in the intracellular/extracellular drug distribution between tumor and normal tissue for drugs exhibiting appropriate pKas.

PO2 in irradiated versus nonirradiated tumors of mice breathing oxygen at normal and elevated pressure.

PURPOSE: To determine if prior tumor irradiation influences tumor pO2 changes in mice breathing oxygen (100%) at normal and elevated pressure. METHODS AND MATERIALS: Single-point pO2 measurements were performed in nonirradiated and previously irradiated (72 h) isotransplanted MCaIV tumors in C3H/Sed mice. Continuous recordings were performed at the same tumor locus under air breathing, followed by 100% oxygen and oxygen at three atmospheres pressure. Following decompression and induction of pentobarbital anesthesia, the procedure was repeated at the same locus. Six nonirradiated and five irradiated tumors were evaluated under the three gas breathing conditions +/- anesthesia. RESULTS: The mean, median, and range of pO2 values did not differ under air-breathing conditions in the nonirradiated vs. previously irradiated tumors. However, prior irradiation substantially enhanced the tumor pO2 increase when the inspired gas phase was switched from air to 100% oxygen at 1 or 3 atmospheres pressure. In four of six nonirradiated tumors, 100% oxygen breathing resulted in a pO2 increase of < 4 mmHg; in the irradiated tumors, the minimum increase was 16 mmHg. Pentobarbital anesthesia did not significantly influence the results obtained. CONCLUSION: These data indicate that the efficacy of oxygen breathing increases during tumor treatment, and suggests that oxygen breathing is a simple nontoxic method for reducing or eliminating radiobiologic hypoxia during therapy.

Quantitative analysis of angiogenesis and growth of bone: effect of indomethacin exposure in a combined in vitro-in vivo approach.

Nonsteroidal anti-inflammatory agents have been used experimentally and clinically to suppress a variety of physiological events, including angiogenesis and formation of bone. The exact mechanisms by which indomethacin alters skeletal tissue generation are unknown, due in part to methodological limitations. By the use of an organ culture assay and an animal model using intravital microscopy in mice bearing dorsal skinfold chambers, the effect of indomethacin on growth and angiogenesis of neonatal femora was characterized over 16 days. In both assays, femora significantly elongated with time (P < 0.05). The in vitro growth rate was more rapid than in vivo and dependent on the serum concentration, culture medium and age of mice. Although enhancing the serum content promoted cellular proliferation in organ culture, it dose-dependently suppressed femoral elongation, leading at 20% fetal calf serum to growth rates identical to those observed in vivo. Indomethacin supplementation (2 and 10 mg l-1) significantly accelerated longitudinal femoral growth in organ culture (P < 0.05), whereas in vivo indomethacin (2 mg kg-1) did not modulate either angiogenesis or elongation of bone. Our in vitro data propose a central role of serum in the regulation of bone formation. Although indomethacin altered femoral growth in vitro, our findings do not suggest that indomethacin suppresses angiogenesis or growth of bone in vivo. The complexity of physiological events in vivo may be obscuring a detectable effect.

Energy status parameters, hypoxia fraction and radiocurability across tumor types.

Under full nutrient in vitro conditions, the cellular adenylate energy charge of six different rodent and human tumor cell types was identical, i.e., 0.94 +/- 0.01, suggesting the potential utility of this parameter as a cell (and tissue) independent marker of nutrient deprivation and hypoxia, across tumor types. The adenylate energy charge values of tumors, arising from these cells, was reduced and variable ranging from 0.72 to 0.91 for the various tumor types. However, neither the tumor adenylate energy charge, NTP/Pi, nor PCr/Pi ratios correlated with the radiobiologic hypoxic cell fractions across tumor types. The reduced adenylate energy charge in vivo suggests varying degrees of nutrient deprivation in the different tumor types, however, factors other than or in addition to hypoxia likely contribute to tumor energy status.

Angiogenesis and growth of isografted bone: quantitative in vivo assay in nude mice.

BACKGROUND: Understanding the regulation of vascularization and formation of bone after skeletal trauma is essential for the development of methods to promote healing. The lack of information on the biology of bone healing led us to establish an experimental model that facilitates the in vivo assessment of angiogenesis and growth of bone. EXPERIMENTAL DESIGN: Fresh, cryopreserved (frozen in the presence or absence of 10% dimethyl sulfoxide (DMSO)) or boiled neonatal femora were transplanted into dorsal skin fold chambers in adult mice of the identical strain, and angiogenesis and growth were monitored over 16 days. Computerized analysis of brightfield and epifluorescence images was employed to characterize the process of angiogenesis. Bone formation was quantified in vivo by the use of oxytetracycline. RESULTS: Reperfusion of pre-existing blood vessels of the graft was observed only in fresh transplanted femora, whereas femora of all experimental groups elicited angiogenic response from the host tissue. The rank order of the angiogenic response was: fresh > cryopreservation with DMSO > cryopreservation without DMSO > boiled. Growth of femora was completely abolished after cryopreservation or boiling. Only fresh transplanted femora increased in length (95 microns/day) and in cartilage diameter (41 microns/day). CONCLUSIONS: Our study demonstrates that (a) angiogenesis and growth of transplanted femora can be chronically assessed using in vivo microscopy; (b) the introduction of oxytetracycline for in vivo fluorescence microscopy allows the differential quantification of bone and cartilage growth; and (c) cryoprotection using DMSO enhances restoration of angiogenic potency after freezing. We consider this assay an excellent experimental model to study in vivo effects of agents or procedures that potentially modulate angiogenesis and growth of bone.

Influence of experimental factors on intrinsic radiosensitivity assays at low doses of radiation: cell multiplicity.

Evaluation of the probability of biological phenomena per cell by colony formation, requires knowledge not only of the number of cells at risk but also of their microdistribution. In the present study, the influence of cellular multiplicity (number of cells per potential colony-forming unit) on the determination of radiation sensitivity is evaluated for a range of multiplicity distributions. Cell surviving fraction was calculated using no multiplicity correction, an average multiplicity correction or the fractional distribution of multiplicities of the control and irradiated population. The results obtained show that: (a) multiplicity corrections are required when the number of cells per potential colony-forming unit is greater than 1.00 either immediately after plating or at the time of irradiation; (b) both the control and irradiated populations must be corrected for multiplicity; (c) multiplicity errors are most pronounced in the low-dose range, e.g. in the survival range with 2 Gy; and (d) the error introduced by using an average vs fractional distribution of multiplicities increases with the multiplicity dispersion. Seemingly small errors due to uncorrected multiplicity effects lead to markedly different predicted isoeffect doses when amplified through multiple (e.g. 30) fractions.

Multivariate determinants of radiocurability. I: Prediction of single fraction tumor control doses.

PURPOSE: The relationship between various laboratory determinants of radiocurability considered alone and in combination, and the observed 50% tumor control dose, has been examined in rodent and xenografted human tumors. METHODS AND MATERIALS: The single fraction 50% tumor control dose (TCD50) under normal and clamp hypoxic conditions, 50% tumor cell transplant dose (Td50), and in vitro estimated tumor cell radiosensitivity parameters, were determined in each of six tumor types (four isografted murine and two xenografted human tumors). Subcutaneous transplant sites and identical or similar tumor generations were used for both the Td50 and TCD50 studies. Radiosensitivity parameters were obtained using the clonogenic assay, after allowing cells to enter the active growth phase to recover from trypsin induced alterations of cell radiosensitivity. Both control and irradiated cells were multiplicity corrected. RESULTS: No single parameter (InTd50, hypoxic fraction, or intrinsic radiosensitivity) correlated with the observed tumor control doses under aerobic or hypoxic conditions. However, when considered in combination, clonogenic fraction (estimated by Td50(-1)), and intrinsic radiosensitivity, predicted the rank-order of tumor control doses with a significant degree of accuracy, and tumor hypoxia influenced the value of the control dose. All parameters were demonstrated to be significant determinants of radiocurability, with substantial tumor to tumor variation in the relative importance of each. For the six tumor types, the combined laboratory determinants predicted 50% tumor control doses which differed from the observed TCD50s by an average of approximately 9 Gy under hypoxic conditions. CONCLUSION: The results obtained demonstrate: (a) the necessity of simultaneously considering all determinants of radiocurability if the role of a single determinant is to be assessed; (b) laboratory determinants may accurately predict tumor radiocurability.

Energy status and radiobiological hypoxia at specified oxygen concentrations.

The relationship between energy status and oxygen concentration was evaluated using Chinese hamster ovary cells over a gas-phase oxygen concentration ranging from 40 to 210,000 ppm O2. Energy status was examined in cells in complete medium equilibrated with specified gas-phase oxygen concentrations, in the presence and absence of glucose. The change in two parameters of energy status, adenylate energy charge, which is ([ATP] + 1/2[ADP])/([ATP] + [ADP] + [AMP]), and ATP/cell as a function of oxygen concentration, was compared with the radiation oxygen enhancement ratio. In the presence of glucose, neither parameter of energy status is substantially affected by oxygen at various concentrations. In the absence of glucose, energy status is affected over the oxygen concentration range which parallels the change in the radiation oxygen enhancement ratio. However, the magnitude of the changes in energy status is dependent upon the duration of oxygen deprivation. In addition, glucose deprivation alone results in a decrease in ATP/cell in the presence of 210,000 ppm O2.

Energy status in the murine FSaII and MCaIV tumors under aerobic and hypoxic conditions: an in-vivo and in-vitro analysis.

The relationship between energy status and hypoxia was examined in two murine tumors with substantially different hypoxic cell fractions in situ and in cells derived from these tumors in vitro. Parameters of tumor energy status were NTP/Pi and PCr/Pi obtained by 31P-NMR spectroscopy and adenylate energy charge and energy status obtained by high-pressure liquid chromatographic analysis of tumor extracts. Adenylate energy charge and rates of high-energy phosphate degradation were determined on cells obtained from both tumor types (MCaIV and FSaII) under identical nutrient and oxygen conditions, that is, air and nitrogen for various durations (0-6 hr). No consistent or substantial differences were noted in the various parameters of tumor energy status obtained by nuclear magnetic resonance analysis or analysis of tumor extracts, even though the MCaIV contains a substantially larger hypoxic fraction (49% vs 12%). Under in vitro conditions, the two cell lines exhibited different responses to oxygen deprivation, the MCaIV being substantially more refractory to energy changes secondary to hypoxia. Noting with caution that this study is based on only two tumor types, our results suggest that differences in cellular capacity for energy maintenance preclude quantitative inferences regarding tumor oxygen status from energy status between tumor types.