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Biomedical subjects

D F Deen

Publications and source records attributed to D F Deen.

At least 91 records · Page 5Linked to original sources
PubMed1984

Combination therapy with 1,3-bis(2-chloroethyl)-1-nitrosourea and low dose rate radiation in the 9L rat brain tumor and spheroid models: implications for brain tumor brachytherapy.

We studied the effects of combination treatment with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and low dose rate radiation in the 9L rat brain tumor in vivo model and the 9L multicellular tumor spheroid model. F-344 rats bearing intracerebral 9L gliosarcomas were implanted with removable 125I sources. Minimal (peripheral) tumor doses of 6387 rad produced an increased life-span (ILS) of 28% over that of control rats implanted with dummy sources, BCNU alone (13.3 mg/kg) produced in an ILS of 67%, and combination treatment with BCNU and implanted 125I sources produced an ILS of 167%. As measured by a colony-forming efficiency assay, the greatest cell kill in 9L spheroids occurred when BCNU was administered 24 hours before irradiation from a 137Cs source at a low dose rate of 5 rad/minute. At a higher dose rate of 210 rad/minute, the time dependence of the effects of combination treatment was identical and therefore independent of dose rate.

Animals↗
PubMed1984

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.

Animals↗
PubMed1984

BCNU-induced sister chromatid exchanges are increased by X irradiation.

We have studied the effect on sister chromatid exchange (SCE) induction in 9L rat brain tumor cells caused by combination treatment with BCNU and X rays. Over the dose and concentration ranges used in these experiments, BCNU induced relatively large numbers of SCEs, while X rays induced few SCEs. When cells were X irradiated immediately after BCNU treatment, the number of SCEs induced was greater than the number of SCEs expected by adding the number of SCEs induced by each agent alone; the number of SCEs induced as a result of this BCNU-X-ray interaction increased as the concentration of BCNU and/or dose of X rays increased. When the addition of bromodeoxyuridine was delayed from 0 to 16 hr after BCNU treatment, the number of SCEs induced declined to control levels by 16 hr. If X irradiation was delayed for up to 16 hr after BCNU treatment the same pattern of decrease was observed; the number of SCEs induced at each time point, however, was greater than that induced by BCNU and X rays alone. X irradiation from 0-16 hr before BCNU treatment produced the same number of SCEs as that produced by BCNU alone. Thus the SCE assay is capable of detecting a drug-X-ray interaction in mammalian cells and provides a sensitive means of studying the sequencing and timing that leads to the interaction.

Animals↗
PubMed1984

The effects of X rays on BCNU-induced DNA crosslinking.

We have used the technique of alkaline elution to study DNA interstrand crosslinking in 9L rat brain tumor cells treated with combinations of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and X rays. Irradiation with doses as low as 50 rad of X rays immediately or 6 hr after a 1-hr treatment with 60, 80, or 100 microM BCNU produced a significant increase in BCNU-induced DNA interstrand crosslinking. If cells were irradiated before BCNU treatment, the amount of crosslinking was not affected compared with BCNU alone. Cell survival experiments using 600 rad of X rays and 1-hr treatments with 0-30 microM BCNU were also performed. As found in the crosslinking studies, irradiation immediately or 6 hr after the BCNU treatment produced enhanced cell kill, but irradiation 6 hr before BCNU treatment did not produce enhanced cell kill. Therefore, the X-ray-mediated increase in BCNU-induced DNA interstrand crosslinking may be the mechanism through which cell kill is increased by combination treatment with the agents.

Animals↗
PubMed1983

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.

Animals↗
PubMed1983

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.

Animals↗
PubMed1983

[Kinetics of multicellular spheroids developed from a human glioblastoma, and effect of 1,3-bis (2-chloroethyl)-1-nitrosourea].

Multicellular spheroids (MCS) were yielded in vitro by early stages of human glioblastoma cell cultures. The spheroids were grown in suspension in the culture medium in agarose-coated (0.65%) dishes. The volume doubling time during the exponential growth phase was longer (13 days) than the population doubling time of monolayer cultures (2-3 days). Pathological examination of MCS showed a thin proliferative outer layer and areas of necrosis in the center. In two experiments BCNU caused a growth delay of MCS which was dose-related. The cells were sensitive to BCNU in monolayer cultures (CFE's assay), whereas the tumor was resistant in vivo. The usefulness and pitfalls of studies using MCS cultured from early passages of human malignant gliomas are discussed.

Brain Neoplasms↗
PubMed1983

The relative resistance of non-cycling cells in 9L multicellular spheroids to spirohydantoin mustard.

Surviving fractions of 9L spheroid cells treated with 1.5, 3.0 and 6.0 micrograms/ml of spirohydantoin mustard (SHM) decreased when assayed 6 hr after treatment but increased thereafter. Flow cytometric analysis showed that exponentially growing 9L monolayer cells treated with SHM accumulated at the G2/M border within 24 hr. Cells dissociated from spheroids treated with 3 and 6.0 micrograms/ml of SHM accumulated at the G2/M border during the first 24 hr after treatment and remained there for the next 12 hr. However, 50% of the cells remained at the 2C DNA peak. Spheroid cells with 2C DNA content 24 hr after treatment were assumed to be non-cycling cells at the time of treatment, and cells that accumulated at the G2/M peak appeared to be cycling cells at the time of treatment; approximately 50% of cells in untreated 9L spheroids are in the noncycling pool. G1 and/or early S phase 9L cells in exponential growth elutriated immediately after treatment with SHM had significantly lower (P greater than 0.001) plating efficiencies than 9L cells in S and G2/M phases. When spheroids were dissociated, elutriated and plated for colony-forming efficiency 24 hr after treatment with 3 micrograms/ml of SHM, fractions enriched in 2C DNA content had significantly higher (P greater than 0.001) plating efficiencies than elutriated cells enriched in 4C DNA. These results indicate that SHM is less effective against non-cycling 9L spheroid cells with 2C DNA content than against cycling 9L spheroids cells.

Animals↗
PubMed1983

Cell cycle age response of 9L cells to 1,3-bis(2-chloroethyl)-1-nitrosourea and modification by alpha-difluoromethylornithine.

We have determined the cell cycle age response of 9L rat brain tumor cells to 1,3-bis(2-chloroethyl)-1-nitrosourea using centrifugal elutriation to obtain populations of cells enriched in G1, S, and G2-M phases. While cells in all phases of the cell cycle were killed by 20 or 40 microM 1,3-bis(2-chloroethyl)-1-nitrosourea, cells in G1 and G2-M phases were more sensitive than cells in S phase. The differential sensitivity was more pronounced at the higher dose, which will markedly alter the distribution of cells through the cell cycle. In a clinical setting, this factor could affect the efficacy of either fractionated or multimodality protocols. Treatment with alpha-difluoromethylornithine, a polyamine biosynthesis inhibitor, potentiated the cytotoxic effects of 20 microM 1,3-bis(2-chloroethyl)-1-nitrosourea against G1- and G2-M- but not against S-phase cells; however, at a higher dose of 1,3-bis(2-chloroethyl)-1-nitrosourea (40 microM), the cytotoxicity was potentiated for cells in all phases of the cell cycle. In alpha-difluoromethylornithine-treated cells, the phenomenon could be reversed by adding 1 mM putrescine 24 hr before treatment with 1,3-bis(2-chloroethyl)-1-nitrosourea. Therefore, the potentiation of 1,3-bis(2-chloroethyl)-1-nitrosourea cytotoxicity appears to be related to polyamine depletion.

Animals↗
PubMed1983

Decreased cytotoxicity of 1-beta-D-arabinofuranosylcytosine in 9L rat brain tumor cells pretreated with alpha-difluoromethylornithine in vitro.

The effect of pretreating 9L rat brain tumor cells in vitro with 10 mM alpha-difluoromethylornithine (DFMO), an enzyme-activated, irreversible inhibitor of ornithine decarboxylase, on the cytotoxicity of 1-beta-D-arabinofuranosylcytosine (ara-C) was investigated using a colony-forming assay. DFMO-mediated polyamine depletion significantly decreased the cytotoxicity of ara-C. The addition of putrescine to DFMO-pretreated cells replenishes intracellular polyamine levels and prevents the decrease in ara-C cytotoxicity. Flow cytometric analysis showed that cells treated with ara-C alone entered S phase semisynchronously within 8 hr after the end of treatment. This phenomenon was virtually eliminated by DFMO pretreatment. Addition of putrescine to DFMO-pretreated cells before treatment with ara-C prevented this DFMO-induced cell cycle kinetic effect.

Animals↗
PubMed1983

Differential potentiation of 1,3-bis(2-chloroethyl)-1-nitrosourea by alpha-difluoromethylornithine in chloroethylnitrosourea-sensitive and -resistant 9L rat brain tumor cells in vitro.

alpha-Difluoromethylornithine, an enzyme-activated, irreversible inhibitor of ornithine decarboxylase, inhibited the growth of both chloroethylnitrosourea-sensitive and -resistant 9L rat brain tumor cells in vitro. After 48 hr of treatment with 10 mM alpha-difluoromethylornithine, the putrescine and spermidine contents of both resistant and sensitive cells were less than 5% of control levels, but the spermine level was slightly elevated. The cytotoxicity of 1,3-bis(2-chloroethyl)-1-nitrosourea, as measured by a colony-forming efficiency assay, was significantly increased in alpha-difluoromethylornithine-pretreated sensitive cells but not in resistant cells treated with this polyamine inhibitor. With the sister chromatid exchange assay, we found that alpha-difluoromethylornithine pretreatment increased 1,3-bis(2-chloroethyl)-1-nitrosourea-induced damage to chromosomes in sensitive but not in resistant cells.

Animals↗
PubMed1983

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.

Animals↗
PubMed1983

Paradoxical behavior of 6-mercaptopurine as a cytotoxic agent: decreasing cell kill with increasing drug dose.

9L rat brain tumor cells were continuously exposed to "low" (2.9 microM) and "high" (18 and 35 microM) doses of 6-mercaptopurine (6-MP); cell kill was measured with a colony forming efficiency assay. The low dose of 6-MP killed more cells than the high doses, as measured by surviving fraction, when they were treated for greater than 48 hours. Even though the total number of cells increased during the 72-hour treatment period, the number of clonogenic cells decreased most when cells were treated with the low dose of 6-MP. Data obtained with flow cytometry suggest that for the high dose there was a drug-induced block of cells in the G1 phase that prevented them from progression into S phase where they would be killed.

Animals↗
PubMed1983

Comparison of the sister chromatid exchange and cell survival assays as a measure of tumor cell sensitivity in vitro to cis-diamminedichloroplatinum (II).

Cytotoxic effects of treating RIF-1, EMT6, and 9L tumor cell lines with cis-diamminedichloroplatinum(II) were measured with the sister chromatid exchange assay and compared to results obtained with the colony-forming efficiency assay. The greatest number of sister chromatid exchanges was induced in RIF-1 cells, fewer in EMT6 cells, and the least in 9L cells. Cell survival data obtained with the colony-forming efficiency assay paralleled data obtained with the sister chromatid exchange assay. These studies suggest that the sister chromatid exchange assay may be a useful method with which to determine the in vitro sensitivity of tumor cells to some antineoplastic agents.

Animals↗
PubMed1983

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.

Animals↗
PubMed1983

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.

Animals↗
PubMed1982

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.

Animals↗