Influence of location within a tumor on cell survival as measured by a clonogenic assay.

In order to elucidate the influence of the location of a cell within a tumor on cell survival, 12 to 16 samples from an individual 9L s.c. tumor were assayed for colony formation. These samples (4 to 6 mg) were taken from untreated, heated, or irradiated tumors, and the variances of the mean relative colony-forming efficiency (RCFE) for individual tumors or groups of similarly treated tumors were compared. These comparisons allowed determination of the effect of location and treatment on the intratumor variability in cell survival. Three factors can influence variability of the RCFE's across tumors: (a) inherent differences in clonogenic capacities; (b) unequal dose distribution of the treatment agent, and (c) different sensitivities of cells to the treatment. Considerable variability was found in the cell survival of control tumors, and treatment of tumors in air-breathing or nitrogen-asphyxiated rats with X-rays did not change the magnitude of this variability. However, the variance of the mean RCFE of heated tumors was an order of magnitude greater than that observed in either control or irradiated tumors. Of the three possible factors that could influence cell survival within a tumor, all were shown to contribute to the variances of the mean RCFE measured in 9L s.c. tumors. However, our study demonstrated that the relative contribution of each of these factors to the variance will probably depend on the treatment agent(s).

Heterogeneity of X-ray cytotoxicity in proliferating and quiescent murine mammary carcinoma cells.

A highly enriched (greater than or equal to 97%) quiescent (Q) tumor cell population can be induced in both the 66 and 67 murine mammary carcinoma lines in vitro by nutrient deprivation (7-day, unfed plateau cultures), while exponential cultures (2-day cultures) of this line are composed of greater than 98% proliferating (P) cells. We have used these two cell lines to determine how the radiation sensitivity varies as a function of genetic heterogeneity (two cell lines derived from the same tumor) and proliferative status (physiological state). The 67 Q cells were significantly more sensitive than were the P cells to single doses of X-rays, with Dos of 52 and 90 rads and Dqs of 188 and 250 rads, respectively. Cells from transition cultures (cells that have essentially stopped proliferation but are not in the biochemical state of Q cells) have a radiation sensitivity similar to that of P cells. When exponentially growing 67 cells were induced into a Q state by reducing the serum concentration (0.5 versus 15%), they, too, were more sensitive to X-rays than were their proliferating counterparts. This sensitivity of the Q cells was decreased by placing them 30 min prior to irradiation in either fresh medium, a balanced salt solution, or a balanced salt solution with 24 mM glucose. However, the Q cells in these conditions were still an order of magnitude more sensitive than the P cells after a 523-rad dose. Therefore, the increased sensitivity of the well-oxygenated 67 Q cells appears to be primarily related to physiological alterations accompanying the transition from P to Q. The radiation sensitivity of 66 cells has also been measured in P and Q states. These cells are significantly more radioresistant than are the 67 cells and, again, the 66 Q cells were more sensitive than were the 66 P cells, with Dos of 90 and 109 rads and Dqs of 150 and 368 rads, respectively. Furthermore, the heterogeneous radiation response of the 66 and 67 cells continues to be expressed under various physiological states, albeit in qualitatively different ways; i.e., in 66 Q versus P cells, the shift in sensitivity is primarily due to a markedly reduced Dq while, in the 67 Q versus P cells, the lowered radiosensitivity is due to a marked reduction in both Do and Dq. At least in these cell lines, it is unlikely that Q cells will determine the response of the tumor to radiation.

Cytotoxic interactions of heat and an ether lipid analogue in human ovarian carcinoma cells.

Ether lipid (EL) analogues of platelet activating factor are known to have a cell membrane-mediated antitumor activity. Although previous studies demonstrated additive interactions with EL and conventional DNA-interacting chemotherapeutic agents, little is known about the interaction of EL with heat. In this study, the cytotoxic interaction of one EL analogue, ET-18-OMe, with heat was measured at two different temperatures, 42 and 44 degrees C, using BG-1 human ovarian carcinoma cells. When the number of colonies, greater than or equal to 40 microns in diameter, was counted as a function of incubation time, the rate of colony formation was suppressed by treatment with ET-18-OMe alone at doses greater than or equal to 2.0 microM or with heat alone. The combination of ET-18-OMe with heat inhibited the colony formation of the slowest growing fraction of the heated cells. The dose-response curve for BG-1 cells after continuous exposure to ET-18-OMe alone was exponential with a small shoulder (Dq = 0.25 microM). The T0 value (the time to reduce survival on the exponential portion of the curve by a factor of 1/e) of the 44 degrees C dose-response curve (30 min) was reduced to half (15 min) by the addition of 0.25 to 1.0 microM ET-18-OMe, but increased again to 24 min when heat was combined with ET-18-OMe concentrations greater than or equal to 2.0 microM. The thermotolerant tail seen in the dose-response curve after continuous heating at 42 degrees C was removed by adding as little as 0.25 microM ET-18-OMe. Isobologram analysis for the combined treatments with 44 degrees C heat and ET-18-OMe at surviving fractions of 0.5, 0.3, 0.1, and 0.01 showed that the treatments were supraadditive at low concentrations (less than 0.5 microM) of ET-18-OMe and additive at moderate concentrations (0.5 to 1.0 microM) of ET-18-OMe. Similarly, the interaction of ET-18-OMe with 42 degrees C heat at surviving fractions of 0.3 and 0.1 was supraadditive at low concentrations (less than 0.5 microM) of the ET-18-OMe and additive with moderate concentrations (0.5 to 1.5 microM) of ET-18-OMe. Because the greatest interaction of ET-18-OMe and heat occurred at clinically achievable doses of both agents, this combination of agents should be considered for use in clinical trials.

Organization of DNA in cerebellar neurons of ageing unirradiated and irradiated rats.

Male Fischer 344 rats were either unirradiated or whole-brain irradiated with single doses of 10.83 or 17.16 Gy of X-rays at 4 months of age, and the organization of the DNA in permanently non-dividing cerebellar neurons examined as a function of age, dose and time after irradiation. In unirradiated rats and rats receiving a whole-brain dose of 10.83 Gy, there were no statistically significant changes in the organization of the bulk DNA and its association with the nuclear matrix as determined by: (a) the sensitivity of the DNA to digestion by micrococcal nuclease, (b) the sensitivity of the nuclear matrix-associated DNA to digestion by DNase I, (c) the relative DNA and protein content of undigested neuronal nuclei, and (d) the relative amount of DNA and protein that is tightly associated with the nuclear matrix after digestion with DNase I. In rats that were irradiated with 17.16 Gy at 4 months of age, there was a gradual decrease in the amount of nuclear proteins as a function of age (P less than 0.003). The amount of protein associated with the nuclear matrix in these irradiated aging rats was also consistently lower than that of their unirradiated counterparts (P less than 0.03). This decrease in the nuclear protein content of the cerebellar neurons in aging rats irradiated with 17.16 Gy may have caused a change in the overall organization of their neuronal DNA. Such a change in the organization of their neuronal DNA was indicated by a higher stainability of their bulk DNA by propidium iodide (P less than 0.03) and a higher sensitivity of the bulk DNA to digestion by m. nuclease (P = 0.087). Although these organizational changes in the neuronal DNA of aging rats irradiated with 17.16 Gy at 4 months of age are subtle, they might alter DNA repair processes or other neuronal functions that may be associated with the "natural" process of aging.

Modification of alkylating agent induced cell kill by 2-nitroimidazoles in unclamped and clamped SC 9L tumors.

Enhanced cell kill has been observed when experimental tumors were treated with alkylating agents in combination with 2-nitroimidazoles (2-NI). In this study, modification of the cell kill induced by cyclophosphamide (CY) and an analog, ifosfamide (IFO), by two radiation sensitizers, misonidazole (MISO) and etanidazole (SR-2508), was measured. Three important parameters were determined: (a) the necessity for hypoxic reduction of the 2-NI to achieve an increase in tumor cell kill, (b) the optimal timing for administration of the alkylating agents and the 2-NI, and (c) the degree of enhancement of the CY- and IFO-induced cell kill. The subcutaneous (sc) 9L tumor model in male Fisher 344 rats was used in these experiments, and the endpoint measured was clonogenic cell survival 18-20 hr after treatment. Under hypoxic conditions, MISO potentiated both CY- and IFO-induced cell kill with a sensitizer enhancement ratio of approximately 1.3 and 1.5, respectively, at the 10(-3) survival level. This enhancement was seen when CY was administered simultaneously or 1.5 hr prior to MISO administration. A similar enhancement of CY-induced cell kill was measured under hypoxic conditions when SR-2508 was used. Enhanced IFO-induced cell kill was measured under hypoxic conditions only when the IFO was given 1 hr before MISO administration. No enhancement of the IFO-induced cell kill was observed when SR-2508 was used instead of MISO. Increased normal tissue damage (i.e., hemorrhagic cystitis) was observed when the MISO was administered along with CY or IFO. Four conclusions can be drawn from these data. Metabolism of the 2-NI by hypoxic cells is necessary for potentiation of CY- or IFO-induced cell kill. Only MISO can potentiate the cell kill induced by IFO. The timing of administration of the alkylating agents and the 2-NI is a critical determinant of the extent of the cell kill obtained. Cell kill induced by IFO appears to be enhanced by MISO to a greater extent than the cell kill induced by CY.

Effect of administration schedules on the potentiation of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) by misonidazole in subcutaneous 9L tumors.

Our previous studies demonstrated that metabolism of misonidazole (MISO) by hypoxic cells is required to potentiate the cytotoxicity of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in sc 9L tumors. To determine the influence of administration schedules on this chemosensitization, tumors were either clamped to produce a reversible hypoxia or left unclamped. MISO (2.5 mmoles kg-1) was administered to rats with unclamped tumors simultaneously with BCNU (9 or 12 mg kg-1), 20 min before BCNU, or 2.5 hr before BCNU, and the drug pharmacokinetics and BCNU cytotoxicity were measured. MISO administered 20 min or 2.5 hr before BCNU increased the plasma elimination half-time (t1/2) of BCNU, but MISO administered simultaneously with BCNU did not change the plasma elimination t1/2 of BCNU. In unclamped sc 9L tumors, all administration schedules decreased the peak BCNU concentration and increased the initial BCNU elimination t1/2; however, the BCNU exposure dose (AUC0-infinity) calculated from these data did not change significantly. In agreement with the AUC calculations, none of the administration schedules altered the BCNU cytotoxicity in unclamped tumors. If the tumors were clamped for 5-120 min after the peak MISO concentration was reached, BCNU-induced cell kill was increased by a constant factor of 3 over the first hour of the clamping period and by an additional factor of 7 over the second hour of the clamping period. If the tumors were clamped for 2 hr after the peak MISO concentration was reached and then BCNU administered 0-60 min after the clamp was released, this chemosensitization remained at a constant factor of approximately 20 for the first 10 min, and then decreased rapidly to a factor of approximately 3 by 20 min after the clamp was released. These data indicate that in sc 9L tumors, (1) at least two biochemical mechanisms are involved in this MISO-BCNU interaction, one of which depends on the duration and extent of the metabolism of MISO by hypoxic cells, and (2) reoxygenation does not immediately eliminate the potentiation of BCNU by MISO. These data also suggest that MISO should be given 2-4 hr before BCNU to achieve the maximum chemosensitization in clinical trials.

Biodistribution of misonidazole and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in rats bearing unclamped and clamped 9L subcutaneous tumors.

The biodistribution of misonidazole (MISO) and 1,3bis(2-chloroethyl)-1-nitrosourea (BCNU) was studied using the subcutaneous (s.c.) 9L tumor model in male Fisher 344 rats. A transient hypoxia in these tumors was created by clamping the blood supply to the tumor. Reoxygenation occurred upon release of the clamp. The plasma and tumor concentrations of MISO and BCNU were quantitated by high pressure liquid chromatography. When 12 mg/kg of BCNU was given i.p. without MISO, the peak plasma concentration was about 6 micrograms/ml, and the elimination half-time was about 16 min. When 2.5 mmole/kg of MISO was given i.p. 150 min before the BCNU, the peak plasma concentration of BCNU increased by approximately 33%, and the plasma elimination half-time increased by approximately 57%. Clamping the tumor for 120 min did not significantly change the BCNU concentration in plasma, but in tumors the time to reach the peak level was delayed slightly, and the peak concentration was reduced when compared to that in the unclamped tumors. MISO pretreatment decreased the BCNU peak concentration in both unclamped and clamped tumors, but the decrease was more pronounced in the unclamped tumors. In both unclamped and clamped tumors, the BCNU concentration and its rate of disappearance were identical about 30 min after BCNU administration, with or without MISO pretreatment. The elimination half-time of MISO from the plasma (approximately 142 min) was identical for rats with unclamped or clamped tumors. The half-time for the disappearance of MISO from unclamped tumors was about 98 min. BCNU had no effect on the MISO concentration in plasma and unclamped tumors. MISO disappeared in the clamped tumors with a half-time of about 40 min. When the clamp was released, the MISO concentration returned to the level in the unclamped tumors after about 45 min. BCNU delayed the return of the MISO concentration to the unclamped tumor level by about 60 min. Two conclusions can be drawn from this study. First, the pharmacokinetics of each drug changed when the two drugs were combined. Second, the data indicate that alterations in the tumor BCNU pharmacokinetics are not the major mechanism responsible for the chemopotentiation previously measured in s.c. 9L tumors.

Chemosensitization of the nitrosoureas by 2-nitroimidazoles in the subcutaneous 9L tumor model: pharmacokinetic and structure-activity considerations.

Alterations of the pharmacokinetics and cytotoxic effects of the nitrosoureas, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and 1-(2-chloroethyl)-3-(cyclohexyl)-1-nitrosourea (CCNU) by the 2-nitroimidazoles, misonidazole (MISO) and SR-2508 were investigated using the subcutaneous (sc) 9L tumor model in male Fisher 344 rats. When 50 mg/kg of CCNU was given i.p., the peak plasma concentration of CCNU was about 3 micrograms/ml. CCNU was eliminated with biphasic kinetics that had a terminal half-time (T1/2) of approximately 47 min. When 2.5 mmole/kg of MISO was given i.p. 150 min before CCNU, the peak plasma concentration of CCNU was increased by approximately 63% with no change in the elimination kinetics. Clamping did not change the pharmacokinetics of CCNU in either plasma or tumors. MISO pretreatment increased the peak CCNU concentration in unclamped tumors by 3-fold, but had no effect on the CCNU pharmacokinetics in clamped tumors. With the exception of a decrease in the peak BCNU concentration in tumors similar to that observed with MISO, SR-2508 (3.75 mmole/kg, i.p.) did not change the pharmacokinetics of BCNU or CCNU in plasma and tumors. CCNU had no effect on the MISO concentration in plasma and unclamped tumors. However, in the clamped tumors, CCNU delayed the return of the MISO concentration to the unclamped tumor level by about an additional 60 min after the clamp was released. SR-2508 was eliminated from the plasma with biphasic kinetics having an initial and terminal T1/2 of approximately 11 and approximately 76 min, respectively. SR-2508 reached a peak tumor concentration of about 500 micrograms/ml in 30 min. The elimination T1/2 for SR-2508 in unclamped and clamped tumors was approximately 81 and approximately 42 min, respectively. When the clamp was released, the SR-2508 concentration returned to the level found in the unclamped tumors approximately 90 min after it reached its nadir; BCNU and CCNU had no effect on the kinetics of this process. MISO significantly potentiated the cytotoxicity of BCNU in clamped tumors at surviving fractions less than or equal to 0.5. MISO did not potentiate the cytotoxicity of CCNU until the surviving fraction reached 0.05. SR-2508 did not potentiate the cytotoxicity of either BCNU or CCNU.(ABSTRACT TRUNCATED AT 400 WORDS)

Feasibility of estimating the temperature distribution in a tumor heated by a waveguide applicator.

The feasibility of using a 2-dimensional (2D) modeling approach for retrospectively describing complete temperature distributions in the midplane of a tumor during a clinical hyperthermia treatment was tested. An experimental treatment, using a 915-MHz waveguide applicator to heat a large melanoma in a dog, was modeled. Detailed measurements of temperatures were made during the treatment. The steady-state blood flow distribution at the midplane was imaged by positron emission tomography (PET), and these data were used to prescribe the modeled perfusion pattern. A 2D finite element method (FEM) was used to approximate the solution to Maxwell's Equations to obtain the specific absorption rate (SAR) distribution. The blood-flow estimates, assumed material properties, SAR distribution, and temperature boundary conditions were then used with the same mesh in a second FEM program to obtain a solution to the bioheat transfer equation. This latter routine was embedded in a state-and-parameter-estimation program that systematically varied selected parameters until the differences between computed and measured temperatures were minimized. Optimizations were performed independently for three subsets of the measured temperature data to assess the sensitivity of the predicted temperature field to the number of measurements. The calculated temperature distributions that resulted were similar to each other, and the predicted temperatures at the sensor points excluded from these optimizations were in reasonable agreement with the measurements. However, lack of unique blood flow values following optimization indicates that the methods of estimating blood flow will need to be improved or that there are problems with model mismatch. This work is a clinical case study of an evolving 2D system of thermal dosimetry which relies on both empirical and theoretical concepts. The methodology is being evaluated for its ability to generate prognostically significant descriptors of the treatment temperature field.

Synthesis and evaluation of novel ether lipid nucleoside conjugates for anti-HIV-1 activity.

Combinations of an amidoalkylphosphocholine, 8, and AZT have been found to cause an apparent synergistic action in suppressing infectious HIV-1 replication. In addition, amidoalkyl, oxyalkyl, and thioalkyl ether lipids have been chemically linked to anti-HIV-1 nucleosides (AZT and DDI) through phosphate and phosphonate linkages. These conjugates have shown promising in vitro anti-HIV-1 activity. Also, the conjugates have a 5-10-fold reduction in cell cytotoxicity compared to AZT alone. The most active compound, an amidoalkyl ether lipid-AZT conjugates, 4A, was found to have a differential selectivity of 1793 in a syncytial plaque assay. In comparison, AZT alone has a value of 1281.

Radiation-induced DNA damage in tumors and normal tissues. III. Oxygen dependence of the formation of strand breaks and DNA-protein crosslinks.

Results from several laboratories, including ours, have suggested that measurements of radiation-induced DNA strand breaks and DNA-protein crosslinks (DPCs) may be used to estimate the hypoxic fraction or fractional hypoxic volume of tumors and normal tissues. This suggestion has been predicted on both published and unpublished information that (1) the oxygen dependence of the formation of strand breaks in irradiated mammalian cells is similar to the oxygen dependence of radiation-induced cell killing, and (2) the oxygen dependence of the formation of DPCs in irradiated mammalian cells is the mirror image of the oxygen dependence of radiation-induced cell killing. However, the published studies that attempted to determine the relationship between the oxygen dependence of the formation of strand breaks and the radiation sensitivity of mammalian cells were not performed at 37 degrees C, the exact oxygen concentrations were not always known, and the results were conflicting. In addition, most of the data on the oxygen dependence of the formation of DPCs are unpublished. Consequently, we have undertaken a comprehensive investigation of one cell line, 9L/Ro rat brain tumor cells, to determine if the shape of the oxygen dependence curve and the Km value for radiation-induced strand breaks and DPCs were similar when 9L cells were irradiated under both ideal gas-liquid equilibrium conditions at 4 degrees C and nonideal gas-liquid equilibrium conditions at 37 degrees C. At 4 degrees C under ideal gas-liquid equilibrium conditions, the Km for the formation of strand breaks was approximately 0.0045 mM, and the Km for radiation sensitivity was approximately 0.005 mM. A similar comparison for the formation of DPCs at 4 degrees C could not be made, because the efficiency of the formation of DPCs was much lower at 4 degrees C than at 37 degrees C. At 37 degrees C under nonideal gas-liquid equilibrium conditions, the apparent Km for the formation of strand breaks and radiation sensitivity was approximately 0.032 mM, and the Km for the formation of DPCs was approximately 0.02 mM. The data for strand breaks are in agreement with the published data of Chapman et al. (Int. J. Radiat. Biol. 26, 383-389, 1974), and the data for DPCs are in agreement with the unpublished data of Meyn (personal communication). These results support the suggestion that measurements of radiation-induced strand breaks and/or DPCs may be used to detect hypoxic cells and estimate the hypoxic fraction or fractional hypoxic volume of tumors and normal tissues.

Cell kill and tumor control after heat treatment with and without vascular occlusion in RIF-1 tumors.

RIF-1 tumors (100-300 mg) were exposed in vivo to heat treatment (41-48 degrees C) for 30 min and then assayed for either cell survival or tumor control. The tumors were heated either with normal perfusion or with temporary vascular occlusion (clamped for 30 min prior to and during the 30-min treatment). The physical technique of water bath heating ensured temperature uniformity in both the perfused and vascularly occluded tumors. Survival curves for tumor cells heated under both conditions had a shoulder and exponential regions. While the T0's were not statistically different in the two cases, cells from the tumors whose blood flow had been occluded showed an enhanced sensitivity to heat as evidenced by a reduction of the shoulder by 2.5 degrees C. A similar increase in sensitivity was measured with the tumor cure assay with the TCT50 decreasing from 47 degrees C for unclamped tumors to 45 degrees C for clamped tumors. The two assays are therefore in excellent agreement in assessing the effectiveness of heat treatment and the influence of vascular occlusion on the heat sensitivity of this tumor. Since the clonogenic assay was performed immediately after treatment, this agreement between assays indicates that direct cell kill by heat is the major factor in determining cure in this tumor.

Alterations of neuronal nuclear matrix and chromatin structure after irradiation under aerobic and anoxic conditions.

This study was undertaken to determine if structural alterations of the bulk chromatin and the amount of protein associated with the nuclear matrix in cerebellar neurons depend on radiation dose and a cell's state of oxygenation. After irradiation with 2.5 to 25.0 Gy under both aerobic and anoxic conditions, the sensitivity of the neuronal chromatin to m. nuclease digestion increase linearly with dose up to about 5 Gy, beyond which there was no further increase. The same increase in accessibility of chromatin to micrococcal nuclease digestion was observed when neuronal nuclei were irradiated at 4 degrees C. Neuronal nuclei were stained with propidium iodide (PI) for DNA and with fluorescein isothiocyanate (FITC) for protein, both before and after complete digestion with DNase I, and analyzed by flow cytometry. There was no change in either the PI (P greater than 0.4) or the FITC (P greater than 0.9) fluorescence of undigested nuclei after irradiation. For the DNase I digested nuclei, the PI fluorescence was unchanged after irradiation (P greater than 0.4), but the FITC fluorescence increased significantly (P less than 0.02). This increase in the FITC fluorescence was linear with dose up to about 5 Gy, beyond which there was no further increase. The flow cytometry results from DNase I digested nuclei were identical for neurons irradiated under aerobic or anoxic conditions, indicating that this phenomenon is oxygen independent. This increase in FITC fluorescence after irradiation was inhibited at ice-cold temperatures and probably reflects an increase in protein content at the nuclear matrix that requires metabolism. This may explain our previously observed resistance of nuclear matrix-associated DNA to digestion by DNase I. This protein increase at the nuclear matrix appears to follow "saturation" kinetics identical to that previously reported for repair of DNA strand breaks in cerebellar neurons. However, the exact molecular nature of this process and its role in DNA repair or cell survival remains to be determined.

Radiation-induced DNA damage in tumors and normal tissues. VI. Estimation of the hypoxic fraction of experimental tumors.

For several years, we have concentrated our efforts on validating the use of radiation-induced DNA strand breaks and DNA-protein crosslinks to assess the oxygenation status of tumors and normal tissues. We have demonstrated that (1) the oxygen dependence of strand break formation is identical to that of radiation-induced cell killing; (2) the oxygen dependence of DNA-protein crosslink formation is the mirror image of that of radiation-induced cell killing; and (3) the formation of these radiation-induced DNA lesions is predominantly dependent on the oxygen concentration near the DNA and is independent of the cell type, metabolic status, proliferative status, pH of the surrounding environment, and composition or properties of the proteins tightly associated with the DNA. In the present study, the hypoxic fraction of three experimental tumors was estimated using our assay of radiation-induced DNA damage. The average hypoxic fraction of a large number of tumors estimated with this assay of radiation-induced DNA damage for (1) WiDR human colorectal carcinoma xenografts (40.8 +/- 4.2%), (2) 66 mouse mammary adenocarcinoma tumors (41.8 +/- 3.1%), and (3) subcutaneous tumors grown from 9L rat brain tumor cells (95% CI =-8.2-4.2%) was not statistically different from that of a large number of tumors measured for each of these tumor models by the paired survival curve method (38.3 +/- 6. 3%, 28.9 +/- 5.5%, 95% CI = 2.2-4.4%, respectively). When the hypoxic fraction measured by the alkaline elution method on one half of an individual tumor was compared to that measured by the paired survival curve method on the other half of the same tumor, no statistical correlation was found for either 66 or WiDR tumors. Although this assay of radiation-induced DNA damage can be used effectively in the laboratory to answer a number of important questions about the oxygenation status of animal tumors and normal tissues, failure to reliably estimate the hypoxic fraction of individual tumors and technical considerations make it unlikely that the assay can be used in the clinic to estimate the hypoxic fraction of human tumors.

Relationship of cell survival, drug dose, and drug uptake after 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine treatment.

The mechanisms that govern the activity and the factors that control the anticancer activity of synthetic ether lipids have not been fully elucidated. In this study, three factors were studied in relationship to cell survival after treatment with 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3): (1) exposure dose, (2) drug uptake, and (3) cell density and cell-cycle distribution. In BG-1 human ovarian carcinoma cells, cell survival was an exponential function of exposure dose and was dependent on drug concentration. Drug uptake was dependent on the concentration of ET-18-OCH3, whereas the reduction in cell survival was directly related to the uptake of drug only in the first decade of cell kill. When the quantity of cells per flask was tripled from 4 to 12 x 10(6) cells, ET-18-OCH3 failed to induce a G2 block. Furthermore, the cell kill induced by a 72-h exposure to 2 microM ET-18-OCH3 was decreased by a factor of 2 when the cell density increased. Therefore, exposure dose and cell density are important parameters in determining the cell kill induced by ET-18-OCH3.

[[(18)F]N-(4'-fluorobenzyl)-4-(3-bromophenyl) acetamide for imaging the sigma receptor status of tumors: comparison with [(18)F]FDG, and [(125)I]IUDR.

A series of biodistribution studies were conducted with the radiotracer, [(18)F]N-(4'-fluorobenzyl)-4-(3-bromophenyl)acetamide, [(18)F]1 in nude mice bearing tumor xenografts of the mouse mammary adenocarcinoma, line 66. This radiotracer has a high affinity for both sigma(1) and sigma(2) receptors. In vivo studies were also conducted in order to assess the effect of blocking sigma(1) receptors on tumor uptake and the tumor:background ratio of this radiotracer. The results of these studies revealed that blocking the sigma(1) receptor so that only the sigma(2) receptors are labeled in vivo, results in a higher tumor:background ratio with only a small reduction in the tumor uptake of the radiotracer relative to the no-carrier-added (i.e., nonselective) conditions. Comparative in vivo studies were also conducted with the anatomic and metabolic imaging agent, [(18)F]FDG, and a radiolabeled DNA precursor, [(125)I]IUdR. Both of these radiolabeled compounds represent classes of agents that have been proposed for imaging the proliferative status of solid tumors. The results of these studies indicated that a sigma(2)-selective imaging agent may be, 1) a better anatomic imaging agent for breast cancer than [(18)F]FDG, and 2) a better functional imaging agent than the radiolabeled DNA precursors, [(123/124)I]IUdR and [(11)C]thymidine, for measuring the proliferative status of breast tumors with PET and SPECT. However, additional studies will be needed to compare sigma(2)-selective imaging agents with [(18)F]FLT in order to determine which is the more appropriate imaging agent for measuring the proliferative status of breast tumors with PET.

Cell kill and cytostasis by ET-18-OCH3 and heat.

BACKGROUND: The ether lipid analogue 1-octadecyl-2-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3) is known to have plasma membrane interacting antitumor activity. However, the mechanism and the mode of action of its activity remain to be elucidated fully. MATERIALS AND METHODS: In this study, the proportion of the inhibitory effects resulting either from direct cell kill or from cytostasis induced by (ET-18-OCH3) alone or in combination with heat in BG-1 human ovarian carcinoma cells was quantitated. RESULTS: The magnitude of both cell kill and cytostasis in BG-1 cells was dependent on the concentration of ET-18-OCH3 and the duration of exposure. Cell kill induced by exposure to 2 or 8 microM ET-18-OCH3 for 1-14 days could be expressed as a simple exponential function of exposure dose (concentration x time). As assayed by colony formation, cell survival after continuous exposure to the drug was significantly reduced from that measured when the drug was removed and the cells were incubated for 14 days in drug-free medium. CONCLUSIONS: We conclude that ET-18-OCH3 induces substantial cytostasis as well as cell kill in BG-1 cells and that this cytostasis is directly proportional to the amount of cell kill induced. These data are important in determining appropriate treatment regimens using ET-18-OCH3, either alone or in combination with other anticancer agents.

Regulation of DNA repair kinetics in proliferative and quiescent tumor cells.

The radiosensitivity and kinetics of repair of radiation-induced DNA damage were determined for proliferative (P) and quiescent (Q) cells of the mouse mammary adenocarcinoma line 67. 67 Q cells are more radiosensitive than 67 P cells. Radiation induced the same amount of DNA damage in both 67 P and 67 Q cells. Both 67 P and 67 Q cells repaired their DNA damage with biphasic kinetics, but the half-times for the fast and slow phase were longer in 67 Q cells. Q cell DNA appeared to be in a more compact or condensed chromatin structure and was less accessible to enzymatic digestion than P cell DNA. These data suggest that 67 Q cells are more sensitive to ionizing radiation than 67 P cells because they repair their radiation-induced DNA damage more slowly, perhaps as a result of their more condensed chromatin structure.