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D F Deen

Publications and source records attributed to D F Deen.

At least 37 records · Page 2Linked to original sources
PubMed1995

Use of pulsed-field gel electrophoresis to investigate factors influencing the measurement of DNA double-strand breaks in human brain tumour specimens.

We used pulsed-field gel electrophoresis combined with densitometry to investigate variables influencing the measurement of DNA double-strand breaks (dsb) in human brain tumour cells and frozen tissue. Our studies showed that this system worked best when we ran the gel at 60 V for 22-25 h at 18 degrees C with a pulse time of 15 s in 1xTris-acetic acid-EDTA buffer. Because the densitometric analysis worked well only when DNA concentrations were within a certain range, we developed a quick assay using a DNA-specific dye (TO-PRO-1) to determine concentrations before making agarose plugs. When we used our optimal procedure to measure dsb in six frozen human brain tumour specimens, we found that radiation-resistant tumours had significantly more initial dsb than did radiation-sensitive tumours. The number of residual dsb could not be ascertained because the process of freezing the specimens destroyed dsb repair ability.

Brain Neoplasms↗
PubMed1994

Design and testing of novel cytotoxic polyamine analogues.

We designed three polyamine analogues, 1,14-diamino-N5-methyl-5,10- diazatetradecane (5me-4-4-4), 1,14-diamino-N5,N5-dimethyl-5,10-diazatetradecane (Q-Amm-4-4-4), and 1,14-bis-(ethylamino)-N5,N5-dimethyl-5,10-diazatetradecane (BE-Q-Amm-4-4-4), on the basis of computer modeling and physical-chemical studies of polyamine-DNA interactions. These analogues differ from natural polyamines and from one another in the charge distribution on their aliphatic backbone. We found that 10 microM 5me-4-4-4 did not inhibit growth and was not cytotoxic to the human brain tumor cell lines SF-767 and SF-126. The same concentrations of Q-Amm-4-4-4 and BE-Q-Amm-4-4-4 inhibited cell growth and killed more than 90% of each cell type on day 7 of the experiment. BE-Q-Amm-4-4-4 was slightly more toxic than Q-Amm-4-4-4 in both cell lines. All three agents either decreased or completely depleted intracellular putrescine and spermidine. Q-Amm-4-4-4 and BE-Q-Amm-4-4-4 each also lowered spermine. The fact that 5me-4-4-4 was nontoxic but that Q-Amm-4-4-4 was cytotoxic and inhibited growth suggests that the charge distribution along the surface of the aliphatic backbone of polyamines is important in determining growth inhibition and cytotoxicity.

Antineoplastic Agents↗
PubMed1994

Radiopotentiation of human brain tumor cells by the spermine analog N1,N14-bis(ethyl)homospermine.

PURPOSE: To determine whether the cytotoxicity produced by radiation can be increased by the spermine analog N1,N14-bis(ethyl)homospermine (BE-4-4-4). METHODS AND MATERIALS: Two human tumor cell lines, SF-126 and U-251 MG, were either treated with 0.1 or 0.4 microM BE-4-4-4 for 3 or 4 days, or with 0.2 microM BE-4-4-4 for 4 days. At the end of BE-4-4-4 treatment, cells were irradiated and assayed immediately. Polyamine levels, cell survival, and cell number were determined. RESULTS: In SF-126 cells, treatment with 0.2 microM BE-4-4-4 for 4 days killed about 50% of the cells and also increased the cytotoxicity of radiation. The dose enhancement ratio was approximately 1.3:1.5, which is similar to that reported for alpha-difluoromethylornithine. Polyamine levels were partially depleted, and growth was inhibited to about 60% of control levels. Pretreatment of cells with either 0.1 or 0.4 microM BE-4-4-4 for 3 or 4 days produced less of an increase in radiation-induced cytotoxicity, even though these exposures killed 30-40% or 60-90% of the cells, respectively. Similar treatment with 0.1-0.4 microM BE-4-4-4 in U-251 MG cells had minimal effects on cytotoxicity and growth inhibition, while treatment with 1.0 microM and 2.0 microM BE-4-4-4 for 4 days produced more than a 50% depletion in polyamine levels and partial inhibition in growth, but failed to demonstrate radiopotentiation. CONCLUSION: The cytotoxic polyamine analog BE-4-4-4 can increase the cytotoxicity caused by radiation in at least one cell line. The amount of potentiation depends on the concentration of the analog, with the most occurring at the intermediate concentration. Because we did not observe potentiation in both cell lines, and because of the dose dependence seen in SF-126 cells, the clinical efficacy produced by combined BE-4-4-4 and radiation protocols may be limited.

Brain Neoplasms↗
PubMed1994

Pretreatment with the polyamine analog 1,19-bis-(ethylamino)-5,10,15-triazanonadecane (BE-4-4-4-4) inhibits etoposide cytotoxicity in U-251 MG (NCI) human brain tumor cells.

We studied whether pretreatment of U-251 MG human brain tumor cells with the polyamine analog 1,19-bis-(ethylamino)-5,10,15-triazanonadecane (BE-4-4-4-4) affected the cytotoxicity of the topoisomerase II inhibitor etoposide. We found that BE-4-4-4-4 protected cells from the cytotoxic effects of etoposide. Possible mechanisms for this protection may be related to enhanced DNA-nuclear matrix association in analog-treated cells.

Brain Neoplasms↗
PubMed1993

Interaction of a polyamine analogue, 1,19-bis-(ethylamino)-5,10,15- triazanonadecane (BE-4-4-4-4), with DNA and effect on growth, survival, and polyamine levels in seven human brain tumor cell lines.

Computer graphics modeling and physicochemical studies of spermine-DNA interactions, as well as experiments in cell culture, indicate that a polyamine analogue with strong affinity for nucleic acids but poor ability to condense and aggregate DNA in vitro should act as an antiproliferative agent if it can enter cells. On the basis of our studies of polyamine-DNA interactions, we designed a pentamine, 1,19-bis(ethylamino)-5,10,15- triazanonadecane (BE-4-4-4-4), that had these characteristics. Measurement of melting temperature and ultraviolet light scattering studies show that the affinity of this analogue for calf-thymus DNA is about 4 times higher than that of spermine, whereas its ability to aggregate DNA is slightly poorer than that of spermine. Studies in U-87 MG, U-251 MG, SF-126, SF-188, SF-763, SF-767, and DAOY human brain tumor cells in tissue culture showed that treatment for more than 96 h with concentrations of 5 microM BE-4-4-4-4 or greater inhibited growth; decreased levels of putrescine, spermidine, and spermine; and decreased colony-forming ability in all cell lines. The cytotoxicity of the analogue varied among cell lines; DAOY and SF-767 were the most sensitive and the most resistant lines, respectively. In SF-763 cells, growth inhibition by BE-4-4-4-4 could be partially reversed by the addition of putrescine, spermidine, or spermine 1 day after BE-4-4-4-4 addition, but in U-251 MG cells, growth inhibition was reversed only by spermine and not by other polyamines. When any of the naturally occurring polyamines was added simultaneously with BE-4-4-4-4, growth inhibition was completely blocked. The data suggest that a threshold intracellular concentration of BE-4-4-4-4 is needed to manifest the growth-inhibitory and cytotoxic effects. In most cell lines, once that threshold level is reached, the growth-inhibitory and cytotoxic properties of the analogue are manifest irrespective of cellular polyamine levels. Further increases in the BE-4-4-4-4 concentration or incubation time reduce the intracellular polyamine levels but do not significantly increase growth inhibition. In U-87 MG and DAOY cells, however, prolonged incubation with higher concentrations of BE-4-4-4-4 causes additional growth inhibition along with depletion of intracellular polyamines.(ABSTRACT TRUNCATED AT 400 WORDS)

Brain Neoplasms↗
PubMed1993

Treatment with a polyamine analog alters DNA-matrix association in HeLa cell nuclei: a nucleoid halo assay.

The polyamine analog 1,14-bis(ethylamino)-5,10-diazatetradecane (BE-4-4-4) depletes polyamines and inhibits the growth of tumor cells in tissue culture. We treated HeLa cells in culture with BE-4-4-4 for different time periods to produce different degrees of polyamine depletion. The cells were lysed and dehistonized to obtain nucleoids containing DNA attached to the nuclear matrix. Titration of the nucleoids with propidium iodide caused an uncoiling of negatively supercoiled DNA, resulting in the formation of a halo surrounding the nucleoid periphery. The halo diameters in both the BE-4-4-4-treated cells and the untreated control cells were measured using a fluorescence image analysis system. As compared to the control cells, the BE-4-4-4-treated cells showed a 20-25% decrease in halo diameter, indicating that there was less relaxation of the negative supercoils in the nuclear DNA of the BE-4-4-4-treated cells than in the controls.

Cell Division↗
PubMed1993

Effects of fractionated radiation therapy on human brain tumor multicellular spheroids.

We investigated the cytotoxic effects of fractionated radiation therapy on multicellular spheroids of human malignant glioma cell lines U-87 MG, U-251 MG, and U-373 MG. Graded doses of x-rays were administered in 1, 3, 8, 15, and 30 fractions over 15 days. The isoeffect dose for a 1 log cell kill ranged from 4-4.5 Gy for a single fraction to 7-8 Gy for an 8-fraction protocol; no additional dose-sparing was achieved with more fractions. Therefore, the effects of individual doses (1.56 Gy) of the 8-fraction protocol were studied in U-251 MG spheroids. A cell survival assay showed that the first dose of radiation killed 30-50% of the cells; subsequent doses usually killed fewer cells. The cell kill after all 8 doses was about 1.0 log. No consistent relationship between the intracellular glutathione level and fraction number was observed. The 24-hour labeling index of the spheroids did not decrease until after the second fraction. Thus, the higher cell kill of the first dose does not seem to be related to cell cycle synchrony. Multinuclear and mononuclear giant cells were limited almost entirely to the periphery of the spheroids and increased with the number of radiation fractions. We conclude that multicellular spheroids can be used to study the biological effects of fractionated radiation therapy on human brain tumor cells. Although this model cannot be used to evaluate the effect of radiation on normal tissue, it may be useful in developing more effective radiation therapy protocols for human brain tumors.

Brain Neoplasms↗
PubMed1993

Cellular resistance to oxidative stress is accompanied by resistance to cisplatin: the significance of increased catalase activity and total glutathione in hydrogen peroxide-resistant fibroblasts.

Studies designed to better understand the involvement of cellular resistance to oxidative stress in mechanisms of cellular resistance to cisplatin were undertaken using H2O2-resistant variants of the HA1 Chinese hamster fibroblast cell line. H2O2-resistant cell lines were resistant to clonogenic inactivation mediated by cisplatin with dose modifying factors at 10% survival of 1.5-3.0, relative to HA1 cells. The most cisplatin resistant of these cell lines (OC5) also demonstrated fewer DNA-DNA crosslinks induced by cisplatin, relative to HA1. Since H2O2-resistant cells contained increased catalase activity as well as total glutathione (GSH) content, the involvement of these cellular antioxidants in the resistance to cisplatin toxicity was evaluated. Treatment of HA1 and H2O2-resistant cell lines (OC5, OC14) with 9 mM aminotriazole reduced catalase activity by 60-65% but had no effect on the cytotoxicity of cisplatin. In contrast, treatment with 5 mM buthionine sulfoximine reduced total GSH by 90% and sensitized the cells to cisplatin cytotoxicity. Furthermore, extracellular reaction of GSH with cisplatin prior to treating HA1 cells reduced the toxicity of the compound, indicating that this reaction is capable of participating in the detoxification of cisplatin. These results indicate that cellular adaptation to oxidative stress renders cells resistant to DNA damage as well as to cytotoxicity associated with cisplatin treatment. Furthermore, increases in total GSH content (but not catalase activity) appear to partially account for cisplatin resistance demonstrated by H2O2-resistant cells.

Animals↗
PubMed1993

The effects of O6-benzylguanine and hypoxia on the cytotoxicity of 1,3-bis(2-chloroethyl)-1-nitrosourea in nitrosourea-resistant SF-763 cells.

O6-Alkylguanine-DNA alkyltransferase (AGT) activity is associated with resistance of brain tumor cell lines to the cytotoxic effects of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). SF-763 cells exhibit high AGT activity and are resistant to BCNU. In this study, we compared the effects of the AGT inhibitor O6-benzylguanine (BG) on the cytotoxicity of BCNU in oxic and hypoxic SF-763 cells; we also measured AGT activity, ornithine decarboxylase (ODC) activity, and polyamine levels to determine if there was any correlation with cell survival as determined by colony-forming efficiency assay. Exponentially growing monolayer cells were pretreated with 10 microM BG for 2 h under oxic or hypoxic (95% nitrogen/5% CO2) conditions and then exposed to graded concentrations of BCNU for 1 h. BG significantly lowered AGT activity but had no cytotoxic effect in oxic or hypoxic cells; hypoxia alone was not cytotoxic. The cytotoxicity of BCNU was 4 times higher in BG-treated hypoxic cells than in oxic cells treated with BCNU alone; the BCNU doses required for a 1-log cell kill were 75 and 300 microM, respectively. ODC activity was lowered by hypoxia alone but was not significantly affected by BG in either hypoxic or oxic cells. Polyamine levels were not significantly affected by hypoxia or BG. These results indicate that pretreatment with BG dramatically lowers AGT activity and increases the cytotoxicity of BCNU in both oxic and hypoxic SF-763 cells. The mechanism of this enhanced cytotoxicity is apparently unrelated to ODC activity or polyamine levels.

Biogenic Polyamines↗
PubMed1992

Production of stable phenotypes from 9L rat brain tumor multicellular spheroids treated with 1,3-bis(2-chloroethyl)-1-nitrosourea.

During chemotherapy and regrowth of brain tumors, tumor-cell heterogeneity, and possibly tumor progression, may change as a result of both the selective forces and mutagenic effects of treatment. We have isolated and characterized drug-response variants of multicellular rat 9L brain-tumor spheroids exposed to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Ten colonies were isolated from spheroids disaggregated immediately after treatment, and 10 colonies were isolated from treated spheroids disaggregated after 1 week in suspension culture. The sensitivity to BCNU was determined by assays of sister chromatid exchange and colony-forming efficiency in monolayer cultures of each subline after a 1-hr exposure to graded doses of BCNU. Three classes of response were found: BCNU sensitivity increased, decreased, or was comparable to that of uncloned, parent 9L cells. Resistant phenotypes were predominant (8/10) in sublines from spheroids disaggregated immediately after treatment, whereas hypersensitive phenotypes (4/8) were isolated only from spheroids disaggregated after 1 week of regrowth. Since subpopulations isolated immediately after treatment do not have the same biological characteristics as those isolated after a period of regrowth, these data suggest that tumor-cell heterogeneity may be generated by distinct processes at various times during therapy. The predominance of hypersensitive sublines obtained by the regrowth protocol may have resulted from the recovery of cells that would have died if isolated but were instead able to repair the drug-induced damage when left in contact with neighboring, possibly resistant cells. Two resistant and two hypersensitive sublines were studied further.

Animals↗
PubMed1991

Effect on N1,N14-bis-(ethyl)-homospermine (BE-4-4-4) on the growth of U-251 MG and SF-188 human brain tumor cells.

We studied the effects of the spermine analogue N1,N14-bis-(ethyl)-homospermine (BE-4-4-4) on growth, survival and polyamine levels in cultured U-251 MG and SF-188 human brain tumor cells. After 48 hr of treatment at concentrations of 1 microM or higher, BE-4-4-4 accumulated in cells with a concomitant decrease in intracellular putrescine, spermidine and spermine concentrations. Growth inhibition by 10 microM BE-4-4-4 began at 6 hr and peaked between 16 and 24 hr. The analogue was also increasingly cytotoxic with doses between 1 and 10 microM and with treatment times between 16 and 48 hr. Polyamines added 1 day after BE-4-4-4 lowered the intracellular concentrations of the analogue but did not reverse its growth-inhibitory activity. When added simultaneously with the analogue, however, polyamines caused a decrease in analogue concentration that was accompanied by a block to the growth inhibition. BE-4-4-4 has a higher affinity for DNA than spermine has, but is less able to aggregate DNA. Its growth-inhibitory and cytotoxic effects support our hypothesis that polyamine analogues that enter cells and replace natural polyamines at DNA binding sites, without fulfilling their biologic functions, should act as antiproliferative agents.

Antineoplastic Agents↗
PubMed1991

Prolonged hyperthermia eliminates mycoplasma from cultured human and rat brain tumor cell lines.

Mycoplasma infection of mammalian cells in culture is a common occurrence that can affect the results of experimental protocols. Current methods of eliminating mycoplasma from cell cultures are usually tedious, time-consuming, and sometimes unsuccessful. In the present study, four cultured brain tumor cell lines (human U-251 MG, U-87 MG, SF-126, and rat 9L) were heavily contaminated with Mycoplasma orale. Heating the cultures to 41 degrees C for at least 96 h eliminated the contamination for up to 7 months, the maximum period of observation. The time chosen to assay for the presence of mycoplasma in cultures was critical: in some cultures heated for less than 96 h that initially appeared to be free of contamination, mycoplasma began to appear after 2 weeks. Heat-treated cells grew at the same rate as unheated control cells. Infected cells were more sensitive to X rays than uncontaminated cells, but the sensitivity reverted to normal after mycoplasma was eliminated by hyperthermia. The heating method does not require a cell cloning procedure or the use of exogenous materials. Treated cell cultures exhibit normal growth and radiation sensitivity, and the technique seems to be reliable and efficient.

Animals↗
PubMed1990

Differential drug sensitivity conferred by growth status detected in a mixed population of cycling and noncycling cells.

The noncycling cell compartment of tumors is considered to be an important target for chemotherapeutic agents; yet, it has been difficult to accurately quantitate its contribution to tumor response because of a lack of methods that can readily discern the relative sensitivities of cycling and noncycling cells. We have used antibodies against bromodeoxyuridine-substituted DNA in a unique experimental protocol that provides a basis for distinguishing the cycling and noncycling cell compartments and detecting their respective levels of drug-induced chromosome damage. A mixed population of cycling and noncycling cells was obtained by culturing 9L rat brain tumor cells as multicellular spheroids. Cell cycle compartments and phase distributions were monitored with flow cytometry using bivariate analysis of DNA content and bromodeoxyuridine incorporation. Bromodeoxyuridine labeling was manipulated to differentially label metaphases from cycling and noncycling cells for sister chromatid exchange. This is based on the differential staining patterns of chromatids and metaphases that are obtained from cells that have replicated in the presence of bromodeoxyuridine. The chromosome damage in each cell cycle compartment following exposure to the chemotherapeutic agent 1,3-bis(2-chloroethyl)-1-nitrosourea was assessed by the number of sister chromatid exchanges induced by treatment. Noncycling cells were shown to be more sensitive to 1,3-bis(2-chloroethyl)-1-nitrosourea-induced damage than were cycling cells. However, when allowed to remain noncycling for 24 h after treatment, the number of exchanges decreased in noncycling cells, which may indicate their ability to recover from damage. These results illustrate an experimental protocol that permits simultaneous assessment of cell cycle compartment recruitment and sister chromatid exchange induction in cells derived from a cytokinetically complex population containing both cycling and noncycling cells.

Animals↗
PubMed1990

Effect of N1,N14-bis(ethyl)homospermine on the growth of U-87 MG and SF-126 human brain tumor cells.

The effect of the spermine analogue N1,N14-bis(ethyl)homospermine on the growth, polyamine levels, and survival of U-87 MG and SF-126 human brain tumor cells was examined in tissue culture. At concentrations of 10 mumols and above, N1,N14-bis(ethyl)homospermine inhibited growth significantly, caused a marked decrease in intracellular levels of the naturally occurring polyamines putrescine, spermidine, and spermine, and had a considerable cytotoxic effect on both cell lines after more than 96 h of treatment. In earlier studies we showed that the affinity of the analogue for calf thymus DNA was higher than the affinity of spermine, but that it did not aggregate DNA or release bound ethidium bromide from DNA as efficiently as spermine does. Therefore, the growth-inhibitory and cytotoxic effects of N1,N14-bis(ethyl)homospermine support our hypothesis that polyamine analogues that can enter cells, deplete intracellular levels of natural polyamines, and replace the natural polyamines from their binding sites on DNA without replacing function should act as antiproliferative agents.

Brain Neoplasms↗
PubMed1990

Effect of alpha-difluoromethylornithine on 1,3-bis(2-chloroethyl)-1-nitrosourea and cis-diamminedichloroplatinum(II) cytotoxicity, DNA interstrand cross-linking, and growth in human brain tumor cell lines in vitro.

The polyamine biosynthesis inhibitor alpha-difluoromethylornithine (DFMO) has been shown to potentiate the cytotoxicity of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in 9L rat brain tumor cells and in non-central nervous system human cancer cells in vitro, but the effects on a human brain tumor cell line have not been reported. Because BCNU is one of the main chemotherapeutic agents used clinically for the treatment of brain tumors, the effect of DFMO treatment on cell growth and potentiation of cytotoxicity was studied in vitro in U-251 MG and SF-126 cells, human tumor cell lines derived from malignant glioma tissue. Pretreatment of U-251 MG with 1 mM DFMO depleted cells of putrescine and spermidine within 48 h but did not sensitize cells to BCNU treatment even after a pretreatment of 72 h. DFMO treatment had no effect on the number of interstrand cross-links formed in BCNU-treated cells. Even treatment with 5 mM DFMO for 72 h caused only the suggestion of potentiation of BCNU cell kill. In contrast, a 72-h pretreatment with 1 mM DFMO decreased the cytotoxic effect of cis-diammine-dichloroplatinum(II) and caused a 38% decrease in the number of DNA interstrand cross-links formed. The glutathione content and cell cycle distribution of U-251 MG cells were not affected by DFMO pretreatment. Because Phase II clinical trials with DFMO and BCNU have shown promise for the treatment of anaplastic astrocytomas in humans, a second brain tumor cell line, SF-126, was studied. In this cell line a consistent potentiation of BCNU cytotoxicity (dose enhancement of 1.2 at the 10% survival level) was observed in cells pretreated with 1 mM DFMO for 72 h.

Biogenic Polyamines↗
PubMed1990

Effect of hypoxia on 1,3-bis(2-chloroethyl)-1-nitrosourea cytotoxicity in 9L cells.

The cytotoxic effects of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) on 9L rat brain tumor cells were studied under oxic and hypoxic conditions. Acute hypoxia was produced by gassing exponentially growing monolayer cells with 95% nitrogen/5% CO2 for 2 h, after which cells were treated with graded concentrations of BCNU for 1 h. Cell survival was assayed with a colony forming-efficiency assay and the extent of DNA cross-linking was measured with the alkaline elution assay. BCNU was more cytotoxic to hypoxic than to oxic cells. There were far more interstrand cross-links formed in hypoxic than in oxic cells, and the number of cross-links could be measured readily in hypoxic cells at very low concentrations of BCNU. This allowed cell survival and cross-linking to be compared at the same dose levels, a correlation not possible for oxic cells in which cross-links are not measurable at low doses. The total intracellular levels of glutathione were lower in hypoxic cells, but the reduced glutathione levels may not be related to the enhanced cell kill because survival of oxic cells treated with BCNU was not affected when cells were depleted of glutathione with buthionine sulfoximine. These results indicate that the additional cell kill produced in 9L cells under hypoxic conditions is related to increased cross-link formation.

Animals↗