Tumor retention of 5-fluorouracil following irradiation observed using 19F nuclear magnetic resonance spectroscopy.

PURPOSE: The combination of 5-fluorouracil (5FU) and radiation results in improved tumor control in a variety of gastrointestinal cancers. We propose the enhancement is related to radiation potentiating the antitumor effects of 5FU. To better understand the mechanism of the 5FU-radiation interaction, 19F nuclear magnetic resonance (NMR) spectroscopy experiments were performed to observed the tumor clearance and metabolism of 5FU. METHODS AND MATERIALS: Experiments were performed on 10 3-6-week-old female (Nu/Nu) athymic nude mice. Flank tumors measuring approximately 1.0 cm in diameter 3 weeks following a subcutaneous injection of 1 x 10(6) human colon adenocarcinoma (HT-29) cells were studied. In our first group, all animals received an intravenous bolus injection of 5FU (100 mg/kg) immediately before spectroscopic analysis. Animals in the second group were first treated with a single tumor radiation dose of 10 Gy just before the 5FU injection and subsequent spectroscopy. Spectroscopic analysis was performed with a 2.0-T NMR spectroscopy system. RESULTS: The tumor retention of 5FU was prolonged in animals receiving radiation before the drug infusion. The tumor clearance rate of the 5FU for nonirradiated animals was 0.0178 +/- 0.0082/min vs. 0.0055 +/- 0.0027/min for irradiated animals, reflecting a threefold reduction in drug clearance in the irradiated tumors. The difference was significant at p < 0.005. CONCLUSION: Our preliminary experiments suggest the enhanced cytotoxicity seen with concurrent 5FU and radiation is related to prolonged tumor retention of 5FU induced by radiation. This is consistent with the hypothesis that radiation is potentiating the cytotoxic effects of 5FU.

Distribution of hypoxia and proliferation associated markers in spontaneous canine tumors.

The therapeutic response of malignant tumors depends on a number of factors associated with tumor microenvironments including the possibility that these microenvironments change during treatment. Two factors, tumor hypoxia and cell proliferation, have been examined in spontaneous canine tumors undergoing multifraction radiation therapy. The approach utilizes immunohistochemical analyses of hypoxia (CCI-103F) and proliferation associated (PCNA) antigens in biopsy samples taken before and after 5 daily fractions of 3 Gy (total dose 15 Gy). The tissue samples were formalin-fixed and paraffin-embedded for the immunohistochemical study. Immunostaining of the sections for PCNA and hypoxia marker reveals little or no overlap when the analysis is made prior to irradiation. An increased degree of overlap seems to occur after 15 Gy but the situation is complicated by a change towards more diffuse PCNA immunostaining in the cells of the irradiated tissues.

The relationship between proliferative and oxygenation status in spontaneous canine tumors.

PURPOSE: Immunocytochemical markers have been applied to biopsy specimens from spontaneous canine tumors to assess the prevalence and spatial distribution of proliferating and hypoxic cells, and their "geographic" relationship to each other. Both types of cells have been implicated in the failure to locally control human tumors treated with radiation and chemotherapy. METHODS AND MATERIALS: For the detection of hypoxic cells, a rabbit polyclonal antibody raised against a protein-bound, hexafluorinated, 2-nitroimidazole, designated CCI-103F, was used. The unmetabolized drug must first be injected into the dog to allow time for hypoxic metabolism and cellular binding to occur. For the detection of proliferating cells, a mouse monoclonal antibody raised against an endogenous nuclear protein, the "proliferating cell nuclear antigen," or PCNA, was used. This protein is expressed in most actively proliferating cells, but not in quiescent ones. An indirect immunostaining technique was used to visualize these markers in the tissue sections, and image analysis was used to estimate the area fraction of positive staining in representative, low magnification microscope fields. RESULTS: Tumors with both high and low hypoxic and proliferative area fractions have been identified. No systematic relationship between the prevalence of the two markers, nor of the relationship between tumor grade and proliferative fraction, could be established. Staining with the proliferation marker was more commonly found near blood vessels, but some "nests" of tumor cells apparently distant from vasculature contained many proliferating cells. Staining with the hypoxia marker tended to be distant from the vasculature and/or bordering regions of tumor necrosis, but some labeled cells appeared near blood vessels, and in the absence of necrosis. Staining of sequential sections, one with the proliferation marker and one with the hypoxia marker, indicated that the two cell populations overlapped to varying extents. Some incidental staining of canine normal tissues with both the proliferative and hypoxia markers was observed as well. CONCLUSION: The immunochemical marker approach promises to be a useful tool to increase both our basic understanding of tumor physiology and the complex nature of tumor heterogeneity.

Description of a spheroid model for the study of radiation and chemotherapy effects on hypoxic tumor cell populations.

The presence of poorly oxygenated cells in solid tumors may account for clinical resistance to ionizing radiation and some chemotherapy in many cancers. Studies of the presence and spatial distribution, sensitivity to cancer therapies, and other physiological characteristics of hypoxic cells are hindered by the lack of markers specific for hypoxia and a relevant yet easily manipulated model system. We have chosen to use multicellular spheroids composed of murine EMT6 (fibrosarcoma) tumor cells as a model system and have applied an immunohistochemical marker specific for hypoxic cells with the ultimate goal of determining how cell populations change in response to radiation and/or chemotherapy. Large spheroids (500-700 microns in diameter) were selected and incubated in the presence of a hexafluorinated 2-nitroimidazole derivative, designated CCI-103F, which undergoes reductive metabolism and irreversibly binds to cellular macromolecules only under low oxygen tensions. A rabbit polyclonal antibody raised against a CCI-103F/protein adduct was used to visualize hypoxic cells using standard streptavidin-biotin-peroxidase immunohistochemical methods. Investigations using this spheroid model system promise to further our understanding of hypoxic cell resistance to cytotoxic therapies and of hypoxic cell biology in general.

Development of an ELISA for the detection of 2-nitroimidazole hypoxia markers bound to tumor tissue.

Canine and rodent tumors covalently bind the fluorinated 2-nitroimidazole, CCI-103F, in a way that immunohistochemical analysis shows is consistent with the location of tumor hypoxia. We have now developed a rapid, quantitative, and non-radioactive enzyme linked immunosorbent assay for the binding of CCI-103F in biopsy samples of spontaneous canine tumors. Issues of antigen stability during tissue processing, calibration of the ELISA, and the use of biopsy samples for measuring tumor hypoxia by the ELISA approach are addressed.

Aerobic radiosensitization by SR 4233 in rodent and human cells: mechanistic and therapeutic implications.

Mammalian cells surviving exposure to the bioreductive, cytotoxic agent SR 4233 under hypoxic conditions are sensitized to X-irradiation under aerobic conditions (and in the absence of drug). Fits of both the single-hit, multi-target and linear-quadratic expressions to survival data, as well as direct measurement of surviving fractions after a dose of 2 Gy, indicate that the aerobic radiosensitization produced by SR 4233 can increase both the initial and final 'slopes' of the X-ray survival curve. The amount of radiosensitization produced, and whether the modification is principally in the slope or shoulder region of the survival curve, varies from cell line to cell line. Rodent cells are radiosensitized equally whether the drug treatment is given immediately before or after, the irradiation, but human cells are only sensitized for SR 4233 exposure administered before irradiation. Using rodent CHO cells, time-course experiments for SR 4233 and X-rays given in sequence, in which an interval of up to 2 h was interposed between the treatments, reveal different kinetics for the loss of radiosensitization depending on whether the hypoxic drug exposure was given before or after the aerobic irradiation. When SR 4233 treatment is given pre-irradiation, the radiosensitization effect persists for at least 2 h, but it does not when drug is given after irradiation. Taken together, the finding of a difference between rodent and human cells with respect to post-irradiation sensitization by SR 4233, and the differing time-course kinetics for this effect as a function of how the drug and radiation are sequenced, suggest that while SR 4233 behaves in a radiomimetic manner in most respects, there may be subtle differences in the nature of the lesions produced by the drug, the important cellular targets for this damage, and/or the cell's management of the damage.

Induction of hypoxia in glass versus Permanox petri dishes.

The survival of Chinese hamster ovary cells in culture following graded doses of X rays delivered under aerobic and hypoxic conditions, or treatment with the bioreductive drug SR 4233 under hypoxic conditions, was evaluated as a function of whether cells were plated onto glass or Permanox plastic petri dishes. In the case of treatment with SR 4233, the influence of varying the total volume of medium in the dishes was also studied. While the Permanox petri dishes were sufficient to yield "radiobiological" hypoxia, that is, oxygen enhancement ratios of approximately 3.0 were obtained for X irradiation, they were inferior to glass petri dishes with respect to the hypoxia-selective cytotoxicity of SR 4233. For a 90-min hypoxic exposure to 40 microM SR 4233, the surviving fraction of cells plated on plastic dishes averaged about 50-fold higher than that of cells plated on glass dishes. Although varying the total medium volume did affect the extent of SR 4233-induced cytotoxicity for glass dishes--drug toxicity decreased slightly with increasing medium volume--this was not the case for the plastic dishes, in which the cell survival following a fixed SR 4233 exposure was essentially constant as a function of medium volume. These results suggest, at least for SR 4233, and under these experimental conditions, that Permanox petri dishes are not satisfactory for such studies.

Aerobic radiosensitization by SR 4233 in vitro and in vivo.

During the past 3 years, our laboratory has identified and characterized the drug SR 4233 (3-amino-1,2,4-benzotriazine 1,4-dioxide) as the lead compound in a series of benzotriazine di-N-oxides that are both potent and selective killers of hypoxic cells in vitro and in rodent tumors in vivo. Recently, we have identified a novel property of SR 4233: the ability of a pre- or post-irradiation drug treatment under hypoxic conditions to radiosensitize aerobic cells in culture. For the mouse cell lines RIF-1 and SCC VII in vitro, this radiosensitization took the form of a steepening of the slope of the acute dose radiation survival curve, although there was also reduced survival in the "shoulder region" of the curve. For both cell lines, the sensitization occurred whether the hypoxic drug exposure was given immediately before or after the irradiation under aerobic conditions. To determine whether radiosensitization could be demonstrated for RIF-1 and SCC VII mouse tumors in vivo, tumor-bearing animals were exposed to 4 daily dose fractions of 5 Gy of X rays either alone, or followed immediately by injections of SR 4233 and the vasoactive agent hydralazine, which increases tumor hypoxia and therefore can potentiate the effect of such hypoxiaspecific drugs. Although treatment with the SR 4233/hydralazine combination after each dose fraction reduced tumor cell survival to between 10(-5) and 10(-6), near the limits of resolution of the clonogenic survival assay, the effect appeared to be strictly additive, suggesting that with this fractionated protocol, aerobic radiosensitization could not be detected. This is likely to be a consequence of the exquisite direct cytotoxicity of the SR 4233 and hydralazine combination toward the hypoxic cells in tumors.

Pre- and post-irradiation radiosensitization by SR 4233.

SR 4233 (3-amino-1,2,4-benzotriazine 1,4-dioxide) is a bioreductive agent which exhibits highly selective killing of hypoxic cells in a variety of mammalian cell lines in vitro and in murine tumors in vivo. The selective toxicity of the drug results from its one-electron reduction under hypoxic conditions to form a free radical intermediate capable of damaging DNA, through the formation of strand breaks. Using the neutral filter elution assay, SR 4233 was found to be more efficient at producing DNA double strand breaks in Chinese hamster ovary (CHO) cells than an equitoxic dose of gamma-rays. Drug and radiation sequencing experiments were also performed, with both cell survival and DNA strand break rejoining used as endpoints. As a result of these studies, we now describe two additional properties of SR 4233: (a) radiosensitization of aerobic cells in culture produced by hypoxic incubation with drug either before or after irradiation, and (b) the inhibition of subsequent rejoining of radiation-induced DNA double strand breaks after hypoxic pretreatment with drug. The magnitude of the radiosensitization produced did not vary for drug treatments which, when given alone, reduced cell survival over a range from 30% to 2%. The extent of DNA repair inhibition increased with increasing severity of the SR 4233 pretreatment, but was quite small for non-lethal drug exposures.

Structure-activity relationships for benzotriazine di-N-oxides.

SR 4233 (3-amino-1,2,4-benzotriazine 1,4-dioxide) is a bioreductive agent that selectively kills and radiosensitizes hypoxic mammalian cells in vitro and murine tumors in vivo. In an attempt to better understand the mechanism of action of the drug, and to determine whether a superior analog may exist, 15 benzotriazine-di-N-oxide analogs of SR 4233 have been evaluated to date for the following properties: hypoxic and aerobic toxicity toward CHO cells in vitro, drug-induced stimulation of oxygen consumption by incubation with respiration-inhibited cells, and acute LD50 evaluated in BALB/c mice. We noted several correlations between these biological properties of the drugs and some of their physicochemical characteristics. Both the hypoxic cytotoxicity and stimulation of oxygen consumption by respiration-inhibited cells were positively correlated with E1/2, the polarographic half-wave reduction potential, and a measure of electron affinity. The air-to-nitrogen differential cytotoxicity reached a maximum (corresponding to SR 4233) and then declined with increasing E1/2. The acute LD50 of each analog in mice decreased with increasing E1/2. One new compound, SR 4482, was found to be more toxic to hypoxic cells in vitro, but less toxic to mice, than SR 4233. It is similar in structure to SR 4233, but lacks any substituent in the 3-position of the triazine ring. This promising drug may represent a member of a new subseries of 1,2,4-benzotriazines with different structure-activity relationships.

Metabolism of SR 4233 by Chinese hamster ovary cells: basis of selective hypoxic cytotoxicity.

The metabolism of SR 4233 (3-amino-1,2,4-bentotriazine-1,4-dioxide), recently reported as highly toxic to hypoxic cells in vitro, was studied by using suspensions of Chinese hamster ovary cells. The rates of formation of two known reduction products, the 1-oxide and the unoxygenated 3-aminobenzotriazine, were measured in aerobic and hypoxic cell suspensions for drug treatments producing both hypoxic and aerobic cytotoxicity. Formation of the 1-oxide and a small amount of the 3-aminobenzotriazine occurred preferentially in hypoxic suspensions. These metabolites were relatively nontoxic to either aerobic or hypoxic cells, implying another mechanism of toxicity. The activation of SR 4233 by single electron transfer, hypothetically forming a toxic drug radical, was explored. Aerobic stimulation of oxygen consumption in respiration-inhibited cells and malondialdehyde release from aerobic cells in the presence of SR 4233 indicated the formation of active oxygen species during drug activation. Increased malondialdehyde release in hypoxic cells and its attenuation by the hydrogen donor, dimethylthiourea, implied the presence of an oxidizing radical. Unlike the nitroimidazole, misonidazole, hypoxic metabolism of SR 4233 did not deplete intracellular glutathione or result in increased binding of drug metabolites to cellular macromolecules. These results are consistent with macromolecular damage caused by an oxygen sensitive, nonbinding, drug-free radical intermediate with oxidizing properties as the mechanism of selective hypoxic toxicity of SR 4233.

Enhancement of radiation-induced tumor cell killing by the hypoxic cell toxin SR 4233.

SR 4233 (3-amino-1,2,4-benzotriazine 1,4-dioxide) is the lead compound in a series of benzotriazine di-N-oxides which exhibit high selective killing of hypoxic mammalian cells in vitro. Drug concentrations to produce equivalent levels of cell killing of SCC VII murine carcinoma cells under hypoxia were nearly 200-fold lower than under aerobic conditions. Following a one hour hypoxic incubation with drug, 20 microM SR 4233 killed 99.9% of SCC VII cells. The hypoxia-specific cytotoxicity of SR 4233 is due to bioreductive metabolism. For in vivo studies, pharmacokinetic measurements showed that drug concentrations well in excess of 20 microM were achievable in SCC VII tumors in mice for approximately one hour after a single injection of SR 4233. Under these conditions, cell killing was considerably enhanced in SCC VII tumors when SR 4233 was combined with a single X-ray dose of 20 Gy. The enhancement was seen whether SR 4233 was given for up to 2 h before or for up to an hour after radiation, and was comparable to the enhanced cell killing achievable with a single large dose of the radiosensitizer misonidazole. While this finding is consistent with the selective killing of at least some subset of hypoxic tumor cells by SR 4233, other interactions between the drug and radiation damage may contribute to the overall effect observed.

SR-4233: a new bioreductive agent with high selective toxicity for hypoxic mammalian cells.

We have examined the effects of the benzotriazine di-N-oxide SR-4233 (3-amino-1,2,4-benzotriazine-1,4 dioxide) on a variety of aerobic and hypoxic cells in culture, and on tumors in mice. The cell lines used were Chinese hamster ovary (HA-1), mouse 10T1/2, RIF-1, and SCC VII cells, and the human cell lines HCT-8, AG1522, and A549. The effect of SR 4233 in combination with irradiation was also examined in the SCC VII tumor growing in the flank of C3H mice using clonogenic assay (tumors excised 24 hr after irradiation). We found SR-4233 to be a potent and selective killer of hypoxic cells. Cell killing as a function of time for the various cell lines was exponential, with no shoulder. Drug concentrations producing equivalent levels of cell killing were 75-200 fold lower in hypoxic than in aerobic cells for the mouse and hamster lines, and 15-50 fold lower for the human cells. In vivo experiments showed that the non-toxic dose of 0.3 mmole/kg of SR-4233 enhanced radiation-induced tumor cell kill when the drug was given between 1 hr before and 2 hr after the radiation dose. We have also shown that the drug metabolizes more rapidly under hypoxic than aerobic conditions, both in vitro and in vivo. The toxic product(s) is unknown, but could be the 1-electron reduction product, the radical anion, because the mono N-oxide (the 2-electron reduction product) did not display cytotoxicity or selective killing under hypoxic conditions. This compound could therefore be a useful tool in tumor biology, as well as being a new lead in the development of bioreductive cytotoxic agents for cancer therapy.

Co-cultured transformed and untransformed C3H 10T1/2 cells: preferential killing of transformed cells by low dose rate irradiation.

Co-cultured C3H 10T1/2 cells, in which transformed cells were grown as discrete colonies on top of density-inhibited monolayers of untransformed cells, were used to determine the potential usefulness of a short term assay system for the study of differential radiation effects as they may apply to cell populations with differing turnover rates, but in close physical contact. Mixed cultures were exposed to either an acute dose of 20 Gy of Cs-137 gamma rays, or a dose of 72 Gy delivered at a low dose rate of 0.34 Gy per hour. These treatments resulted in approximately equal levels of damage to the untransformed monolayers. At 10-day intervals after treatment, representative flasks from each dose group were examined for evidence of degeneration, and subsequent regrowth, if any, of the transformed colonies and untransformed monolayers. For comparable amounts of visible damage to the untransformed monolayers, the low dose rate irradiation was more effective at delaying regrowth of, or even eradicating, transformed colonies. These results are consistent with expectations based on previous results in which dose-rate or dose fractionation isoeffect curves were compared for these two cell types, grown independently in plateau phase cultures.

Loss of repair capacity in density-inhibited cultures of C3H 10T1/2 cells during multifraction irradiation.

Multifraction survival curves for slowly cycling, density-inhibited C3H 10T1/2 cells were shown previously to bend toward lower survival levels with increasing total dose, even for doses per fraction as small as about 2.0 Gy. In an attempt to explain this, we tested the capacity of cells to repair potentially lethal damage (PLD) as fractionation progressed. Plateau-phase cultures were exposed to repeated doses of 4.0 Gy of 137Cs gamma rays delivered at 12-hr intervals. After zero, three, five, and seven fractions, some cultures were put aside, incubated for 12 hr at 37 degrees C, irradiated with a single dose of 9.0 Gy, and subsequently returned to a 37 degrees C incubator. At 0, 2, 4, 6, and 12 hr after the 9.0 Gy dose, cultures were trypsinized and plated for a survival assay. Following three fractions of 4.0 Gy, cells were able to repair PLD as well as those receiving a single dose of 9.0 Gy without prior fractionation. Following five fractions, cells were less able to repair PLD, and after seven fractions, only a very small amount of PLD repair was detectable using this method of measurement.

Patterns of cell loss and repopulation in irradiated cultures of plateau phase C3H 10T 1/2 cells.

Patterns of cell loss and repopulation were studied in plateau phase cultures of slowly-cycling, contact-inhibited C3H 10T1/2 mouse fibroblasts following large single, and multiple small doses of 137Cs-gamma rays. A progressive, dose-independent cell loss was apparent within days after irradiation with large single doses, and similar patterns of loss were observed following the start of multifraction irradiations. This progressive cell loss culminated in the loss of integrity of the monolayer of cells, a loss of contact-inhibition, and therefore, an increased rate of cell division. The time of onset of measurable repopulation, and the time required to completely repopulate a culture varied with the number of clonogenic cells present at the time of breakdown of the monolayer. For cultures receiving multiple irradiations over time periods greater than those required for breakdown of the monolayer, repopulation began to occur during treatment, and its rate varied with the average dose per fraction being delivered. Thus, repopulation did not start immediately after the start of irradiation, but needed a triggering event, in this case, a decrease to a critical level in the cell density. Once initiated, repopulation was able to decrease or even eliminate the effectiveness of subsequent doses in reducing the number of viable cells per culture. To the extent that the responses of slowly-cycling, contact-inhibited cells in vitro can be applied to interpret the radiation responses of cell populations in vivo, these results further support the notion that it may be necessary, in some cases, to account for an increasing contribution from repopulation with increasing overall treatment time in dose fractionation isoeffect formulae used for predicting tissue tolerances or tumor control.

Dose fractionation effects in plateau-phase cultures of C3H 10T1/2 cells and their transformed counterparts.

A comparison of gamma-ray dose fractionation effects was made using plateau-phase cultures of C3H 10T1/2 cells and their transformed counterparts in an attempt to simulate basically similar populations of cells that differ primarily in their turnover rates. The status of cell populations with respect to their turnover rates may be an important factor influencing dose fractionation effects in early- and late-responding tissues. In this cell culture system, the rate of cell turnover was approximately three times higher for the plateau-phase transformed cultures. While the single acute dose survival curves for log-phase cells were indistinguishable, there were significant differences between the survival curves for plateau-phase cultures of the two cell types. These differences were qualitatively similar to the differences recently postulated for the survival of target cells governing early and late tissue responses. Both cell lines had a similar capacity for repair of sublethal damage, but untransformed cells had a much greater capacity to repair potentially lethal damage in plateau phase. Further, untransformed plateau-phase cultures were much more sensitive to a radiation-induced G1 (or G0 to G1) delay than transformed cultures. Multifraction survival curves were determined for both cell lines for doses per fraction ranging from 9.0 to 0.8 Gy, and from these isoeffect curves of log total dose versus dose per fraction were derived. The isoeffect curve for the slowly cycling, untransformed cells was found to be appreciably steeper than that for the more rapidly cycling transformed cells, a finding consistent with previously reported differences in dose fractionation isoeffect curves for early- and late-responding tissues in vivo.

Dose-rate effects in mammalian cells: V. Dose fractionation effects in noncycling C3H 10T 1/2 cells.

Multiple gamma ray dose fractionation effects were studied using contact-inhibited C3H 10T 1/2 murine fibroblast cultures in an attempt to simulate conditions in tissues with low rates of cell turnover. Multifraction survival curves were determined for different doses per fraction using 12 hour interfraction intervals 7 days per week. Isoeffect curves were generated from the multifraction survival curves. For doses per fraction greater than approximately 1.0 Gy, these isoeffect curves were similar to those derived from tissue reactions in humans and experimental animals published previously. For doses per fraction below 1.0 Gy, the isoeffect curves became essentially flat, thus deviating appreciably from the total dose which would be predicted by extrapolating the NSD equation to achieve an isoeffect. The existence of a non-repairable component of gamma ray damage can be inferred from this finding, which has implications both for basic radiobiology, and radiotherapy.

Changes in early and late effects with dose-per-fraction: alpha, beta, redistribution and repair.

Both tumor response and the manifestation of "early" and "late" effects in normal tissues as a function of overall treatment time and dose per fraction are thought to reflect an interplay between the inherent sensitivity of the target cells at risk, and their proliferation kinetics. In light of clinical and experimental studies which indicate that differences exist between early and late radiation responses in normal tissues after changes in fractionation pattern, several investigators have postulated that the intrinsic sensitivities of the respective target cells, in particular the alpha/beta ratios, are different. We suggest that the clinical and experimental findings may be explained, at least partly, in terms of cell cycle redistribution, and may also involve changes in the ability of early effects target cells to repair potentially lethal damage.

Differential responses to x-irradiation of subpopulations of two heterogeneous human carcinomas in vitro.

The responses of two heterogeneous human cancer cell lines and their derivative clones to graded single doses of X-rays were examined in vitro. One system consisted of the human colon carcinoma line DLD-1 and two subpopulations (clones A and D). The second system consisted of the human lung carcinoma line (LX1) and four subpopulations (LX1-1, LX1-2, LX1-3, and LX1-9). These subpopulations have previously been shown to be markedly heterogeneous in terms of such characteristics as karyotype, morphology, drug sensitivity, tumorigenicity, and expression of membrane glycoproteins (such as carcinoembryonic antigen and tumor colonic mucoprotein antigen). Exponentially growing cultures were irradiated with graded single doses of 100-kVp X-rays. Survival was assessed using colony formation as the end point, and responses from multiple experiments were fitted to the single-hit, multitarget equation of cell survival. Values for the mean lethal dose (D0, grays), quasithreshold dose (Dq, grays), and extrapolation number (n) were obtained. For the human colon adenocarcinoma system, these values for the three tumor lines were: DLD-1, 0.95, 2.34, and 11.7; clone A, 1.06, 2.23 and 8.20; and clone D, 1.08, 1.89, and 5.80. For the human lung carcinoma system, these values for the five sublines were: LX1, 1.14, 0.19, and 1.20; LX1-1, 0.96, 2.06, and 8.54; LX1-2, 0.98, 0.88, and 2.48; LX1-3, 0.68, 2.05, and 20.3; and LX1-9, 1.12, 0.00, and 1.00. These two human tumor systems therefore exhibit variability in their intrinsic sensitivity to X-irradiation. The data indicate that failure of some human carcinomas to respond to physical treatment modalities can be due to preexisting resistant subpopulations.