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Biomedical subjects

D F Deen

Publications and source records attributed to D F Deen.

At least 73 records · Page 4Linked to original sources
PubMed1985

Potentiation of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)-induced cytotoxicity in 9L cells by pretreatment with 6-thioguanine.

9L Rat brain tumor cells were treated with 0.2 microM 6-thioguanine for 48 hr, which produced a 40% cell kill, a small (15%) inhibition of cell growth, and an accumulation of cells in S-phase. Maximum incorporation of [14C]6-thioguanine into cellular DNA occurred after 24 hr of incubation; 70% of the label was incorporated into DNA as 6-thio-2'-deoxyguanosine. Pretreatment of 9L cells for 48 hr with 0.2 microM 6-thioguanine potentiated the cytotoxicity of 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) by 50% with a dose enhancement ratio of 1.5, and caused a 30% increase in the number of BCNU-induced sister chromatid exchanges (SCEs) and a 50% increase in DNA crosslinks formed, compared to treatment with BCNU alone. Used as a single agent, 6-thioguanine induced a significant number of SCEs. Results suggest that these effects may be related to the increased formation of DNA crosslinks, possibly as the result of the formation of S6-(2-chloroethyl)-6-thioguanine in cellular DNA.

Animals↗
PubMed1985

The effect of treatment with single and split doses of spirohydantoin mustard on the growth delay of 9L rat brain tumor multicellular spheroids.

The effects of single and split doses of spirohydantoin mustard on the growth delay and cell survival of 9L rat brain tumor multicellular spheroids have been investigated. Treatment with 10-25 microM concentrations of spirohydantoin mustard inhibited the growth of spheroids. Growth delay increased rapidly when assayed during the first 6-10 days after treatment, after which a decrease in delay was observed. Plots of the values for the inflection points from growth curves vs drug concentration were linear, and growth delay correlated well with cell survival. There was less growth delay caused by treating spheroids with two 10 microM concentrations of spirohydantoin mustard than with a single 20 microM concentration. When a 2 or 4 hr time interval was allowed between treatment with 10 microM concentrations of spirohydantoin mustard the delay observed was greater than that obtained with the split dose control. When the resolution of the growth delay assay is considered, the split dose effect can be explained by the existence of a 5 microM threshold before any growth delay is observed. Nonetheless, the finding that spheroids treated on the split dose protocol recover from drug damage may have implications for the design of clinical protocols.

Animals↗
PubMed1985

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.

Animals↗
PubMed1985

Comparative study of the effects of hyperthermia and BCNU on BCNU-sensitive and BCNU-resistant 9L rat brain tumor cells.

1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU)-sensitive and BCNU-resistant 9L rat brain tumor cells were treated with BCNU at graded temperatures between 37 and 44 degrees C. The cytotoxic effects of hyperthermia alone on both cell lines were the same. Treating both cell lines with BCNU at temperatures above 37 degrees C caused a progressive increase in cell kill. All survival curves for drug-sensitive cells had shoulders followed by a region of exponential cell kill; dose enhancement ratios calculated at the 10% survival level ranged from 1.7 to 3.0. Survival curves for drug-resistant cells were exponential without a shoulder; dose enhancement ratios ranged from 3.3 to 8.4. For each cell line, a similar amount of the increased cell kill could be explained on the basis of the increases concentration of reactive species produced by hydrolysis of BCNU at elevated temperatures. The amount of cell kill that cannot be explained on this basis, however, suggests that factors other than an increase in the concentration of reactive species at higher temperatures are involved in the enhanced cell killing. Possible mechanisms include a heat-induced change in the structure of DNA chromatin and the effect of isocyanate deactivation of repair enzymes, both of which could lead to an increase in the number of crosslinks formed and therefore to an increase in cytotoxicity.

Animals↗
PubMed1985

Effects of hyperthermia on DNA interstrand crosslinking after treatment with BCNU in 9L rat brain tumor cells.

The effects of hyperthermia (42 degrees C) on 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)-mediated DNA interstrand crosslink formation were investigated in 9L rat brain tumor cells using the technique of alkaline elution. When cells were treated with 60 microM BCNU for 1 hr at 37 degrees C and incubated for 6 hr in drug-free medium at 42 degrees C, there was a 50% increase in crosslinking; and when cells were treated at 42 degrees C and incubated at 37 degrees C, there was a 45% increase in crosslinking compared with the results for cells treated and incubated at 37 degrees C. When cells were treated and incubated at 42 degrees C, there was a 129% increase in DNA crosslinking. The same relative order of results was found for cell survival. These results suggest that hyperthermia can increase DNA interstrand crosslink formation and the consequent cell death through two independent mechanisms: an increase in the amount of initial alkylation because of the increased rate of hydrolysis of BCNU at higher temperatures, and the effect of heat on DNA structure that leads to an increase in the number of crosslinks formed.

Animals↗
PubMed1985

Growth delay in 9L rat brain tumor spheroids after irradiation with single and split doses of X rays.

The response of 9L spheroids to irradiation with single and split doses of X rays has been investigated. Irradiation with single doses caused a dose-dependent decrease in spheroid growth rate, which eventually returned to the growth rate for unirradiated spheroids. This delay appeared to be related to cell survival. When spheroids were irradiated with two 4-Gy doses of X rays separated by various times the amount of growth delay was intermediate between that observed with single doses of 4 and 8 Gy. For relatively short times (15-90 min), recovery probably resulted from repair processes, but for longer times (up to 24 hr), recovery also appeared to depend on cellular redistribution and repopulation effects.

Animals↗
PubMed1984

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.

Animals↗
PubMed1984

Prediction of the relative in vitro sensitivity of 9L rat brain tumor cells to nitrosoureas by the sister chromatid exchange assay.

In an earlier study we showed that there is a good correlation between sister chromatid exchange induction and cell kill in 9L cells treated with certain nitrosoureas. In the study reported here, we treated four 9L cell lines that have different sensitivities to chloroethylnitrosoureas with 1,3-bis (2-chloroethyl)-1-nitrosourea, chlorozotocin, and ethylnitrosourea and determined the number of sister chromatid exchanges induced. Cell lines that were most sensitive to the drugs with respect to cell kill were also most sensitive to induction of sister chromatid exchanges for a given drug, and the assay based on sister chromatid exchange is therefore predictive of the relative sensitivity of these cells to the drugs used.

Animals↗
PubMed1984

Potentiation of 1,3-bis(2-chloroethyl)-1-nitrosourea cytotoxicity in 9L rat brain tumor cells by methylglyoxal-bis(guanylhydrazone), an inhibitor of S-adenosyl-L-methionine decarboxylase.

Methylglyoxal-bis(guanylhydrazone) (MGBG), a potent inhibitor of the spermidine and spermine biosynthetic enzyme S-adenosyl-L-methionine decarboxylase, enhanced the cytotoxicity of 1,3-bis-(2-chlorethyl)-1-nitrosourea in 9L rat brain tumor cells in vitro, as measured by a colony-forming efficiency assay, by an amount that was approximately the same as the potentiation caused by the ornithine decarboxylase inhibitor alpha-difluoromethylornithine. Dose enhancement ratios at 10, 1 and 0.1% survival levels were approximately 1.3 for both inhibitors. 9L cells that were treated for 48 hr with 40 microM MGBG had putrescine, spermidine and spermine levels that were 112, 41 and 21%, respectively, of polyamine levels in control cells. MGBG treatment does not increase intracellular levels of decarboxylated S-adenosyl-L-methionine (AdoMet) as alpha-difluoromethylornithine treatment does. Elevated levels of decarboxylated AdoMet could modify intracellular methylation reactions and could affect the cytotoxicity of a chloroethylnitrosourea. Despite the fact that MGBG treatment caused a slight increase in intracellular levels of AdoMet, it is unlikely that this elevation will increase the amount of intracellular methylation. Thus it appears that effects caused by the decrease in polyamine levels are responsible for the potentiation of chloroethylnitrosourea cytotoxicity against 9L cells.

Adenosylmethionine Decarboxylase↗
PubMed1984

Detection of heterogeneity in the chemosensitivity of 9L brain tumor cell lines to 1,3-bis (2-chloroethyl)-1-nitrosourea by the sister chromatid exchange assay.

To investigate the potential use of the sister chromatid exchange assay to analyze the heterogeneity of drug response among tumor cell subpopulations, mixtures of 1,3-bis (2-chloroethyl)-1-nitrosourea-sensitive (9L) and -resistant (9L2) rat brain tumor cells were treated in vitro with 2 microM 1,3-bis (2-chloroethyl)-1-nitrosourea. When data were plotted as histograms representing the number of cells vs sister chromatid exchanges/metaphase, two regions corresponding to the 9L and 9L2 populations were obtained. The approximate percentages of 9L and 9L2 in each mixture could be predicted from these histograms. While these results were obtained with a limited model chosen for its simplicity, they suggest that the sister chromatid exchange assay may be useful in the analysis of heterogeneity in drug sensitivity among cell subpopulations in a tumor.

Animals↗
PubMed1984

Possible factors in the potentiation of 1-(2-chloroethyl)-3-trans-4-methylcyclohexyl-1-nitrosourea cytotoxicity by alpha-difluoromethylornithine in 9L rat brain tumor cells.

Depletion of intracellular levels of polyamines in 9L rat brain tumor cells by alpha-difluoromethylornithine (DFMO), an enzyme-activated irreversible inhibitor of ornithine decarboxylase, significantly enhanced the cytotoxicity of 1-(2-chloroethyl)-3-trans-4-methylcyclohexyl-1-nitrosourea (MeCCNU) in vitro as measured by a colony-forming efficiency assay. Administered as a single agent, DFMO was not cytotoxic to 9L cells. Treatment for 48 hr with 10, 1, 0.5 or 0.1 mM DFMO produced similar levels of polyamine depletion-and similar potentiation of MeCCNU cytotoxicity. Restoration of intracellular polyamine levels by the addition of exogenous putrescine (1 mM) to treated cells prevented the potentiation of MeCCNU, which indicates that this phenomenon might be the result of polyamine depletion. DNA adduct formation in polyamine-depleted and control cells was studied with [14C]-MeCCNU; no difference in monoadduct formation was found between polyamine-depleted and control cells. Experiments to determine whether polyamine depletion has an effect on enzymes involved in the repair of alkylated bases showed that the activity of O6-methylguanine-DNA demethylase, 7-methylguanine-DNA glycosylase and 3-methyladenine-DNA glycosylase were unaffected by 48 hr of treatment with 10 mM DFMO. DFMO treatment causes a substantial increase in the intracellular content of decarboxylated S-adenosyl-L-methionine, which was reversed by addition of putrescine. The possibility that the elevation of decarboxylated S-adenosyl-L-methionine rather than the depletion of polyamines is responsible for the effects of DFMO is discussed.

Animals↗
PubMed1984

Relative biological effectiveness of 125I sources in a murine brachytherapy model.

The relative biological effectiveness of 125I and 192Ir has been determined in a murine brachytherapy model that uses a clonogenic cell assay as the end point. Removable 125I or 192Ir sources were implanted at right angles to the surface of RIF-1 tumors grown in the flanks of C3H/He mice. After irradiation for 1-5 days, mice were sacrificed and isodosed annuli of irradiated tumor tissue were sampled for the clonogenic cell assay. The slopes and intercepts of the two radiation survival curves for implanted sources with activities of 10 to 50 Gy for 125I (dose rate = 39.8 +/- 4.3 cGy/hr) and 192Ir (dose rate = 42.3 +/- 2.7 cGy/hr) were identical; the relative biological effectiveness was 1.

Animals↗
PubMed1984

The effect of combination treatment with cis-platinum and low dose rate 125I radiation in a murine brachytherapy model.

We studied the effect of combination treatment with cis-platinum during low dose rate irradiation from 125I sources in the RIF-1 murine flank tumor model. Cell survival measured with a colony forming efficiency assay was used as the experimental endpoint. Sources implanted into tumors delivered 25 Gy of radiation to isodosed annuli of tissue at a dose rate of 1.3 Gy/hr. When 3 mg/kg of cis-platinum was administered 24, 12, and 1 hr before or 0, 12 and 24 hr after irradiation, the effect on cell kill was additive. Thus in the RIF-1 model there is no particular advantage gained by treatment with the combinations of cis-platinum and 125I radiation used in these studies.

Animals↗
PubMed1984

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.

Animals↗
PubMed1984

Effects of alpha-difluoromethylornithine on the growth of 9L rat brain tumor multicellular spheroids and their response to 1,3-bis(2-chloroethyl)-1-nitrosourea.

9L rat brain tumor cell spheroids were treated with alpha-difluoromethylornithine (DFMO) and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) alone and in combination. In contrast to results obtained with 9L monolayer culture cells, very low concentrations of DFMO killed spheroid cell within 0.5 day after the start of treatment; cell kill was maximum within 2 to 3 days. DFMO cytotoxicity could be prevented by adding putrescine (to a final concentration of 1 mM) to the culture medium. DFMO also significantly slowed and eventually stopped the growth of spheroids in a dose-dependent manner. Cells in all regions of DFMO-pretreated spheroids were sensitized to BCNU as measured by colony-forming efficiency; this sensitization was prevented when putrescine was added to culture medium before BCNU treatment. When used as single agents, either a 3-day treatment with 10 mM DFMO or a 1-hr treatment with BCNU (1.5 micrograms/ml) produced similar growth delay, but used in combination, the two agents produced a much longer growth delay than produced by either agent alone. Growth of spheroids treated continuously for up to 28 days with 10 mM DFMO ceased at approximately 7 times the volume at the time of treatment. When spheroid cells were treated for 1 hr with BCNU (1.5 micrograms/ml) and then were treated continuously with DFMO, growth plateaued at approximately 3.5 times the volume at the time of treatment; when spheroids were treated first with DFMO for 3 days, then with BCNU (1.5 micrograms/ml) for 1 hr, and then treated continuously with DFMO, growth plateaued at approximately 1.5 times the volume at the time of treatment. The number of clonogenic cells per spheroid that survived combination treatment also reflected the cytotoxic effects of the two drugs. Thus, the combined drug treatment was very effective in inhibiting the growth of spheroids and in preventing an increase in the number of clonogenic cells per spheroid.

Animals↗
PubMed1984

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.

Animals↗
PubMed1984

Decreased cell kill of vincristine and methotrexate against 9L rat brain tumor cells in vitro caused by alpha-difluoromethylornithine-induced polyamine depletion.

The effect of polyamine depletion on the cell kill caused by the cell cycle-specific agents vincristine (VCR) and methotrexate (MTX) was studied in 9L rat brain tumor cells in vitro using a colony-forming efficiency assay as the experimental end point. The cell kill produced by a 24-hr treatment with VCR or MTX was decreased in 9L cells pretreated with 1 mM alpha-difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase. Reversal of the alpha-difluoromethylornithine-induced polyamine depletion with 1 mM exogenous putrescine prevented the decrease in VCR and MTX cytotoxicity. After a 48-hr treatment with 1 mM alpha-difluoromethylornithine, the number of mitotic cells in asynchronously growing 9L cell cultures was reduced markedly. The decreased cell kill of VCR and MTX appeared to be the result of polyamine depletion-induced inhibition of 9L cell cycle traverse, which reduced the number of cells in drug-sensitive phases of the cell cycle and thereby reduced the cell kill caused by the drugs.

Animals↗