Effect of cell-cell interactions on drug sensitivity and growth of drug-sensitive and -resistant tumor cells in spheroids.

Multicellular spheroids were grown from mixtures of rat brain tumor cells sensitive (9L) and resistant (R3) to 1,3-bis(2-chloroethyl)-1-nitrosourea. Percentages of each cell subpopulation in these spheroids were estimated with the sister chromatid exchange assay and were found to be approximately the same as those used to initiate spheroids. Spheroids grown from 9L cells alone had a higher growth rate than spheroids grown from R3 cells alone. However, the growth rate of mixed-cell spheroids was essentially the same as that of pure 9L spheroids and was independent of the percentages of R3 cells in mixed-cell spheroids. The sensitivity of 9L cells in mixed-cell spheroids treated with 1,3-bis(2-chloroethyl)-1-nitrosourea, estimated by changes in the number of sister chromatid exchanges per metaphase induced by treatment, decreased as the percentage of R3 cells increased. These effects are probably the result of an interaction between the two cell subpopulations held in three-dimensional contact, a situation similar to that in tumors in situ. The results suggest why one cell subpopulation of tumors does not become dominant during growth and indicate that interactions between cell subpopulations can influence the sensitivity of one subpopulation to 1,3-bis(2-chloroethyl)-1-nitrosourea.

alpha-Difluoromethylornithine-induced polyamine depletion of 9L tumor cells modifies drug-induced DNA cross-link formation.

Depletion of intracellular levels of polyamines, which are believed to have a role in the intranuclear stabilization of DNA, alters the cytotoxicity of 1,3-bis(2-chloroethyl)-1-nitrosourea and cis-diamminedichloroplatinum II in 9L rat brain tumor cells. Alkaline elution techniques were used to show that polyamine depletion alters the number of DNA cross-links formed by these cytotoxic agents.

Polyamine depletion influences drug-induced chromosomal damage.

Polyamines have been implicated in the intracellular stabilization of DNA. Depletion of intracellular polyamines influences the cytotoxicity of 1,3-bis(2-chloroethyl)-1-nitrosourea and cis-diamminedichloroplatinum II. By means of the sister chromatid exchange assay, it was found that intracellular polyamine depletion can also alter the induction of chromosomal damage by these cytotoxic agents.

Depletion of intracellular polyamines may alter DNA conformation in 9L rat brain tumor cells.

Depletion of polyamines in 9L rat brain tumor cells by treatment with alpha-difluoromethylornithine dramatically altered DNA conformation as measured by viscoelastometry. The reduction of intracellular putrescine and spermidine concentrations to less than 5 percent of their concentrations in control cells decreased the sensitivity of 9L cell DNA to x-irradiation and increased the maximum viscoelastic retardation time of the DNA. Both of these phenomena were reversed by addition of exogenous putrescine.

Quantitative dose-response relations for the cytotoxic activity of chloroethylnitrosoureas in cell culture.

A dose-response relation for the cytotoxic activity of chloroethylnitrosourea cancer chemotherapeutic agents in cell culture has been developed. Data for the activity of 1,3-bis(2-chloroethyl)-1-nitrosourea, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea, and 1-(2-chloroethyl)-3-(piperidine-3,6-dion-3-yl)-1-nitrosourea against 9L rat brain tumor cells are presented. Cytotoxicity resulting from treatment schedules at different initial drug concentrations, exposure periods, and preincubation periods are correlated using a proposed dose function. Equations are presented that incorporate drug concentration, duration of exposure of cells, and the rate constant for conversion of the drug from an active intermediate into a single dose function. When compared in this way, all cell treatment schedules used were found to be equally effective in killing cells in culture. The cytotoxic activity of the four different chloroethyl-nitrosourea analogs were also found to be the same. These data demonstrate the application of quantitative dose-response relations to the activity of chloroethylnitrosoureas in cell culture and provide new insight into the mechanism of action and structure-activity relationships of these compounds.

Effect of feeder cell-released substances on the survival of clonogenic 9L cells after treatment with antimetabolites.

Exponentially growing monolayer cultures of 9L rat brain tumor cells were treated with either 5-fluorouracil or methotrexate. The surviving fraction of cells was determined by a colony formation assay. After 5-fluorouracil treatment, 2 to 5 X 10(5) feeder cells were required to maximize surviving fractions for each drug concentration and to generate a biphasic dose-response curve. If only 1 X 10(4) feeder cells were used, the dose-response curve was steep. Uridine added to the dishes containing 1 X 10(4) feeder cells restored the biphasic shape, while uridine and thymidine added to the dishes yielded the same curve obtained with 2 X 10(5) feeder cells. After methotrexate treatment, the surviving fraction of cells was dependent on feeder cell number when the medium in the dishes was supplemented with dialyzed fetal bovine serum, but it was not dependent on feeder cell number when nondialyzed fetal bovine serum was used. Biphasic dose-response curves were generated from methotrexate-treated cells plated in medium supplemented with either dialyzed or nondialyzed serum, but the drug was more toxic to cells plated in medium containing dialyzed serum. This additional toxicity could be reduced if either thymidine or N-5-formyltetrahydrofolate were added to the dishes and eliminated if 1 X 10(4) feeders were added. These results suggest that any cell culture system used to evaluate antimetabolites should be optimized for possible feeder cell and serum effects.

Double elution analysis of cross-link formation and repair in cycling and noncycling 9L spheroid cells.

We developed the double elution analysis technique, a modification of standard alkaline elution techniques, to assess cross-link formation and repair in the cycling and noncycling cell populations of multicellular spheroids. DNA cross-linking from cycling cells was measured using radioisotope incorporation; DNA cross-linking from the total cell population was estimated using a fluorometric method. Cross-link formation in the noncycling spheroid cells was calculated from these DNA measurements and the percentage of cycling cells as determined by autoradiography. The isotopic and fluorometric assays yielded equivalent elution profiles in cells irradiated with an X-ray dose of 6 Gy, and DNA interstrand cross-link formation by nitrogen mustard was equivalent in the cycling and the total cell population. The rate of cross-link removal appeared to be faster, however, in the cycling cell population. Double elution analysis should be applicable for the investigation of a variety of antineoplastic drugs that produce cross-links and for measuring damage to cycling and noncycling cell populations in tumors from experimental animals, as well as in multicellular spheroid tumor models.

Factors that influence initiation and growth of 9L rat brain gliosarcoma multicellular spheroids.

This report details optimum conditions for initiating and growing 9L rat brain gliosarcoma multicellular spheroids. Best results were achieved when 0.5 to 2.0 x 10(6) cells were seeded initially into agarose (0.5 to 1.0%)-coated Petri dishes. Spheroids initiated in this manner appeared to grow in spinner culture at a rate slightly faster than spheroids initiated and grown in spinner culture. An optimum growth rate was obtained for individual spheroids grown in the well of cluster dishes coated with 0.5 to 0.75% agarose in growth medium containing 10% newborn calf serum and 0.9 g glucose per liter. Three methods for dissociation of spheroids at both room temperature and 37 degrees have been developed. The "race track" method of fractional dissociation produced a constant and similar number of cells for each disaggregation interval, and the plating efficiency of cells in each fraction was similar.

Measurement of radiation-induced damage in human glioma cells with flow cytometry.

Using flow cytometry, we studied DNA supercoiling changes in human glioma cell line SF-126 after irradiation. To release nucleoids (dehistonized DNA in a supercoiled form attached to the nuclear matrix), cells were lysed in a high-salt buffer. Radiation-induced changes in nucleoids were measured by flow cytometry as changes in forward light scatter. Propidium iodide titration curves showed that rewinding of DNA supercoils in irradiated cells was inhibited. To optimize the experimental conditions, we analyzed the effect of lysis time and nucleoid size distribution within the sample. Under optimal conditions, changes in nucleoids were detected after radiation doses as low as 0.5 Gy. The repair of radiation-induced damage in nucleoids followed biphasic kinetics; 50% of the damage was repaired within about 5 min, and the remainder within about 30 min. Interestingly, irradiated S-phase cells showed less damage, as measured by this assay, than irradiated G1- or G2-phase cells, which is consistent with the relative radioresistance of S-phase cells as measured with cell survival assays. Our findings show that flow cytometric measurement of supercoiling changes is a sensitive and relatively rapid method for quantitating radiation-induced damage in individual cells.

Effects of dicyclohexylamine sulfate, a spermidine synthase inhibitor, in 9L rat brain tumor cells.

Growth characteristics, polyamine levels, and distribution of cells in the cell cycle were determined for 9L rat brain tumor cells treated for various periods with 1 mM dicyclohexylamine sulfate (DCHA). Continuous treatment of cells with DCHA caused growth inhibition at 2 days of treatment. After 2 days of treatment the growth rate of cells increased to approximately the same rate as control cells, even though treatment was continuous. Levels of spermidine were depleted to less than 10% of control levels, spermine levels were essentially unchanged, and putrescine levels were elevated to more than 350% of control levels after 9L cells were treated with DCHA for 2 days. In contrast to results found for the polyamine biosynthesis inhibitor alpha-difluoromethylornithine, treatment of 9L cells with DCHA did not potentiate the cytotoxicity of 1,3-bis(2-chloroethyl)-1-nitrosourea. To mimic the effects on polyamine levels caused by treatment with DCHA, 9L cells were treated with 5 mM putrescine alone or with 5 mM putrescine and 1 mM DCHA after treatment with 1 mM alpha-difluoromethylornithine. Results of these experiments suggest that treatment with DCHA alone does not potentiate the cytotoxicity of 1,3-bis(2-chloroethyl)-1-nitrosourea because elevated levels of putrescine caused by treatment counteract the effects of decreased spermidine levels.

Influence of polyamine depletion caused by alpha-difluoromethylornithine, an enzyme-activated irreversible inhibitor of ornithine decarboxylase, on alkylation- and carbamoylation-induced cytotoxicity in 9L rat brain tumor cells in vitro.

Using a colony-forming efficiency assay, we studied the effect of polyamine depletion on the cytotoxicity of four nitrosoureas with different capacities to alkylate and/or carbamoylate biomolecules. 9L rat brain tumor cells were treated with 10 mM alpha-difluoromethylornithine for 48 hr before a 1-hr treatment with nitrosoureas. The cytotoxicity of 2-[3-(2-chloroethyl)-3-nitrosoureido]-D-glucopyranose, which alkylates and subsequently cross-links DNA but does not carbamoylate, was significantly increased by depletion of intracellular polyamines; the dose enhancement ratio of 1.3 is identical to that found for 1,3-bis(2-chloroethyl)-1-nitrosourea and 1-(2-chloroethyl)-3-trans-4-methylcyclohexyl-1-nitrosourea in previous studies. Addition of exogenous putrescine to polyamine-depleted 9L cells 24 hr before treatment prevented this phenomenon. In contrast, the cytotoxicity of 1,3-bis(trans-4-hydroxycyclohexyl)-1-nitrosourea, which carbamoylates only, was significantly decreased in polyamine-depleted cells. This compound alone reduced intracellular polyamine levels. Polyamine depletion did not affect the cytotoxicity of the monoalkylating nitrosoureas N-ethyl-N-nitrosourea and N-methyl-N-nitrosourea. Thus, polyamine depletion apparently potentiates the cytotoxicity only of chloroethylnitrosoureas that alkylate and cross-link.

Nitrosourea-induced sister chromatid exchanges and correlation to cell survival in 9L rat brain tumor cells.

The ability of various nitrosoureas to induce sister chromatid exchanges (SCEs) in 9L rat brain tumor cells was investigated. Treatment of cells for 1 hr with the alkylating and cross-linking agents 1,3-bis(2-chloroethyl)-1-nitrosourea or chlorozotocin produced concentration-dependent increases in SCEs; elevations above controls were detected at concentrations of 1 microM or more. Above 0.25 mM, the alkylating agent ethylnitrosourea produced a dose-dependent increase in SCEs. Treatment with the carbamoylating agent 1,3-bis(trans-4-hydroxycyclohexyl)-1-nitrosourea did not induce SCEs. The maximum drug concentration at which SCEs are readily scored kills approximately 50% of cells. When accurate cell survival data in this dose range were obtained, a direct correlation between nitrosourea-induced cell kill, measured by a colony-forming efficiency assay, and SCE induction was found. Thus, analysis of the levels of SCE production may provide information about the efficacy of antineoplastic drugs.

Cytokinetic and cytotoxic effects of antimetabolites on 9L rat brain tumor cells in vitro.

Treatment of exponentially growing 9L monolayer cells with graded concentrations of methotrexate or 5-fluorouracil (5-FUra) resulted in approximately 95% cell kill, corresponding to the percentage of cells in this system labeled by autoradiography (98%). Additional cell kill could be obtained if 5-FUra was administered at a high concentration (7.7 x 10(-4) M) and could be partly reversed in the presence of 8.2 x 10(-5) M uridine. When cell kill was measured as a function of time of exposure, a 3-hr exposure to 2.2 x 10(-4) M methotrexate or 7.7 x 10(-6) or 10(-4) M 5-FUra killed 30 to 50% of the cell population, which is equivalent to the percentage of the S-phase cells of the system. With both agents, additional cell kill was offset by division of unaffected G2 cells and a drug-induced block of G1 cells at the G1-S border, and it depended on long drug exposure. Nontoxic concentrations of 5-FUra could synchronize and increase the fraction of cells in the drug-sensitive S phase.

Decreased cytotoxicity of cis-diamminedichloroplatinum(II) by alpha-difluoromethylornithine depletion of polyamines in 9L rat brain tumor cells in vitro.

Before treatment with the cytotoxic drug cis-dichlorodiammineplatinum(II) (cis-platinum), 9L rat brain gliosarcoma cells grown in vitro were depleted of intracellular polyamines by alpha-difluoromethylornithine (DFMO; 10 mM, 48 hr), an enzyme-activated, irreversible inhibitor of the polyamine-synthetic enzyme ornithine decarboxylase. In contrast to studies that showed that the cytotoxicity of 1,3-bis (2-chloroethyl)-1-nitrosourea as measured by colony-forming efficiency is enhanced by DFMO depletion of intracellular polyamines, the cytotoxicity of cis-platinum was significantly decreased in polyamine-depleted 9L cells. At 10, 1, and 0.1% survival levels, the dose modification factors were 2.0 to 2.1. Addition of exogenous putrescine to polyamine-depleted 9L cells 24 hr before treatment with cis-platinum partially reversed this phenomenon. When 9L cells were treated either with DFMO and cis-platinum or with DFMO, putrescine, and cis-platinum for 1 hr, a time period that is too short for DFMO to affect intracellular polyamine levels, the cytotoxicity of cis-platinum was decreased, but to a significantly lesser extent than by the 48-hr DFMO pretreatment. Putrescine alone did not alter the cytotoxic effect of cis-platinum when administered either 24 or 1 hr before treatment. DFMO appears to affect cis-platinum cytotoxicity by two different mechanisms, the first mediated through polyamine depletion and the second through a direct interaction with cis-platinum.

Changes in the glutathione content of rat 9L cells induced by treatment with the ornithine decarboxylase inhibitor alpha-difluoromethylornithine.

Treatment of 9L cells with the ornithine decarboxylase inhibitor alpha-difluoromethylornithine (DFMO) depletes cells of putrescine and spermidine and sensitizes cells to the cytotoxic effects of the alkylating agent 1,3-bis(2-chloroethyl)-1-nitrosourea. Because depletion of intracellular levels of the sulfhydryl compound glutathione also increases the cytotoxicity of alkylating agents, the effect of DFMO on intracellular glutathione content was investigated to determine whether DFMO-induced sensitization could be explained by a sulfhydryl-related mechanism. Treatment of 9L cells with 1 mM DFMO caused no change in the glutathione concentration within 48 h; sensitization of cells to 1,3-bis(2-chloroethyl)-1-nitrosourea is generally studied after 48 h of treatment with DFMO. Incubation of cells with DFMO for longer periods caused an increase in glutathione that is related to the depletion of polyamines and not to a decrease in growth rate or a cell cycle effect. Increased glutathione content is not due to a decrease in glutathione catabolism, but rather to an apparent increase in the rate of synthesis of the tripeptide.

Response of 9L rat brain tumor multicellular spheroids to single and fractionated doses of 1,3-bis(2-chloroethyl)-1-nitrosourea.

This study was designed to examine the relative effect of each of four fractions of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) against 9L rat brain tumor multicellular spheroids and to compare the results of the cell survival and growth delay assays. Similar levels of cell kill resulted when BCNU was administered either as single fractions of 1.5, 3.0, 4.5, or 6.0 micrograms/ml for 1 hr or as one to four fractions of 1.5 micrograms/ml that were administered sequentially for 1 hr each. Survival was increased if the assay was delayed until 24 hr after drug treatment, which indicates that 9L cells in spheroids recover from BCNU-induced potentially lethal damage. When BCNU was administered in 1.5-micrograms/ml fractions, plating efficiencies depended markedly on the interval between the fractions. The 12-hr protocol produced an overall higher cell kill. Fractionation schedules of 24 and 36 hr produced less cell kill than did the other schedules. Survival plateaued for the last three treatments with BCNU in the 36-hr schedule. Cells in S phase at the time of administration of the initial 1.5-micrograms/ml fraction of BCNU moved into G1- and G2-M phases by 12 hr after treatment. For time periods longer than 12 hr, cells began to appear in the BCNU-resistant S phase. Thus, the movement of cells into the drug-sensitive and -resistant phases after the first fraction correlates well with the corresponding overall cytotoxic effect produced by treatment with the combined BCNU (1.5 micrograms/ml) fractions. For a higher concentration (3.0 micrograms/ml for 1 hr), maximum cell kill was reached within the 12- to 18-hr interval, after which cell kill plateaued. Cells were not found in the S-phase fraction 12 to 36 hr after the first treatment with 3.0 micrograms/ml; maximum cell kill for the fractionated protocols resulted at these times. Therefore, BCNU, which is classified as a cell cycle-nonspecific drug, can induce a partial synchrony in 9L spheroid cells, which determines the overall cytotoxicity produced by fractionated BCNU protocols. Although spheroids did not shrink during or after exposure to BCNU, growth was retarded by treatment with all doses and schedules. An optimum time point for growth delay measurement could not be determined from the data. However, correlations between cell survival and growth delay were obtained with arbitrarily chosen end point volumes of four and ten times the volume at the time of treatment.

Decreased cytotoxicity of aziridinylbenzoquinone caused by polyamine depletion in 9L rat brain tumor cells in vitro.

The in vitro cytotoxicity of aziridinylbenzoquinone (AZQ) used either alone or after induced intracellular polyamine depletion in 9L rat brain tumor cells was studied using a colony-forming efficiency assay. Used alone, AZQ was cytotoxic to 9L cells; however, depletion of intracellular putrescine and spermidine levels by treatment with 1 mM alpha-difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase, for 72 hr decreased significantly the cytotoxicity of AZQ. Dose modification factors were 1.9 and 1.8 at 10 and 1% survival levels, respectively. Decreased cytotoxicity could be almost completely prevented by addition of putrescine to polyamine-depleted cells 24 hr before AZQ treatment. Although AZQ alone was cytotoxic against 9L cells, metabolic activation by the S-9 rat liver microsomal fraction increased greatly the observed cytotoxicity. However, even with microsomal activation, pretreatment of cells with 1 mM alpha-difluoromethylornithine for 48 hr produced a significant decrease in AZQ cytotoxicity; dose modification factors were 2.4 and 2.2 at 10 and 1% survival levels, respectively. Addition of putrescine to polyamine-depleted cells 24 hr before AZQ treatment prevented the decrease in cytotoxicity. Pretreatment of 9L cells for 48 hr with 40 microM methylglyoxal bis(guanylhydrazone), a polyamine biosynthesis inhibitor that competitively inhibits S-adenosylmethionine decarboxylase, caused a decrease in the cytotoxicity of AZQ administered without microsomal activation. The dose modification factor was 1.6 at both 10 and 1% survival levels.

Time dependence of the potentiation of 1,3-bis(2-chloroethyl)-1-nitrosourea cytotoxicity caused by alpha-difluoromethylornithine-induced polyamine depletion in 9L rat brain tumor cells.

Treatment of 9L rat brain tumor cells with 1.0 mM alpha-difluoromethylornithine (DFMO) produced a time-dependent depletion of cellular putrescine and spermidine. An increase in the potentiation of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) cytotoxicity, measured with a colony-forming efficiency assay, followed the time course of polyamine depletion, reaching its maximum at 48 hr, the time at which maximum polyamine depletion was achieved. Treatment with DFMO at concentrations as low as 0.05 mM for 48 hr effectively depleted putrescine and spermidine and potentiated BCNU cytotoxicity. Treatment for 96 hr with 0.01 mM DFMO produced a partial decrease in putrescine and spermidine levels and a moderate potentiation of BCNU cytotoxicity. The amount of polyamine depletion in 9L cells treated with 0.05, 0.1, and 0.5 mM DFMO was identical at both 48 and 96 hr, but potentiation of BCNU cytotoxicity was greater at 96 hr than at 48 hr. When 9L cells were treated for 48 hr with 1 mM DFMO and DFMO was then removed from the cultures, polyamine levels did not reach control levels by 96 hr after change of medium. The potentiation of BCNU cytotoxicity during this 96-hr period correlated with the extent of polyamine depletion. When 100 microM putrescine was added to the culture medium after DFMO pretreatment (1 mM), polyamine levels approached those of control cells by 24 hr, and the amount of potentiation of DFMO cytotoxicity decreased. These results show that potentiation of BCNU cytotoxicity correlates closely with the amount of DFMO-induced polyamine depletion in 9L cells.

Effects of extreme hypoxia on the growth and viability of EMT6/SF mouse tumor cells in vitro.

The purpose of this study was to characterize a model system in which to study hypoxic cell biology in vitro as a function of time under extremely hypoxic conditions. EMT6/SF cells that were maintained at 37 degrees under hypoxic conditions showed no increase in cell number for up to 70 hr. The mitotic index of hypoxic cultures was less than 0.1%, compared to 2.3 to 3.0% in aerated cultures. The plating efficiency of hypoxic cells decreased with time to 20 to 30% of control values by 70 hr. Aerated cultures consumed glucose more rapidly than did hypoxic ones, due to increasing cell number in air. But, on a per cell basis, hypoxic and aerated cells consumed glucose at equal rates (congruent to 1.2 X 10(-4) micrograms/cell/hr). Virtually 100% of the glucose consumed was converted into lactic acid in both aerated and hypoxic cultures. The labeling index and rate of incorporation of [3H]thymidine decreased exponentially with time in hypoxia. However, the percentage of cells with S-phase DNA content remained nearly constant for up to 72 hr. The rate of protein synthesis was suppressed in hypoxic cultures to between 20 and 50% of control (aerated) rates. When cultures were reaerated following 45 hr of hypoxia, congruent to 12 hr was required for resumption of DNA synthesis and cell division. The application of this system to further study of hypoxic cell biology is discussed.

Sensitization of 9L rat brain gliosarcoma cells to 1,3-bis(2-chloroethyl)-1-nitrosourea by alpha-difluoromethylornithine, an ornithine decarboxylase inhibitor.

alpha-Difluoromethylornithine, a known inhibitor of polyamine biosynthesis, significantly enhanced the cytotoxic effect of 1,3-bis(2-chloroethyl)-1-nitrosourea, a cell cycle-nonspecific agent, in 9L rat brain gliosarcoma cells in vitro. Administered as a single agent, alpha-difluoromethylornithine was not cytotoxic to 9L cells and, compared to untreated control cells, caused no perturbation of cell cycle kinetics. alpha-Difluoromethylornithine-induced depletion of intracellular polyamine levels appears to have caused the observed sensitization of 9L cells to 1,3-bis(2-chloroethyl)-1-nitrosourea. Restoration of intracellular polyamine levels by the addition of exogenous putrescine to treated 9L cells reversed this phenomenon.