Search

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.

The ability of the in vitro sister chromatid exchange (SCE) assay to predict in vivo tumor drug sensitivity was investigated using a spontaneous hepatocarcinoma in C3Hf/Kam mice and 3 chemotherapeutic agents: melphalan; cis-platinum; and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). For hepatocarcinoma cells grown in monolayer culture, melphalan was the most efficient at inducing SCEs, and BCNU, the least. cis-Platinum induced a range in SCEs that overlapped those of BCNU and melphalan, suggesting that hepatocarcinoma is not a homogeneous population with intermediate sensitivity, but is a mixture of cis-platinum-sensitive and -resistant cells. According to in vitro cell survival curves, hepatocarcinoma was most sensitive to melphalan, less sensitive to cis-platinum, and essentially resistant to BCNU. The relative antineoplastic effects of melphalan, cis-platinum, and BCNU in vivo were compared by the response of artificial and spontaneous pulmonary metastases and solid tumors to these agents. For artificial metastases, there was a dose-dependent decrease in the number of lung nodules in mice treated with melphalan or cis-platinum, with melphalan being the more effective. BCNU had no effect. Spontaneous pulmonary metastases generated from hepatocarcinoma leg tumors were reduced in those mice treated with melphalan, unaffected by cis-platinum, and increased by BCNU. In hepatocarcinoma leg tumors (5 to 6 mm in diameter), melphalan induced the longest growth delay, and BCNU the least. Therefore, the relative effects produced by these three drugs in vivo were the same as predicted by SCE induction in vitro. The SCE assay may thus have potential clinical application.

It has been suggested that terminally differentiated mammalian cells have a decreased DNA repair capacity, compared with proliferating stem cells. To investigate this hypothesis, we have examined gamma-ray-induced DNA strand breaks and their repair in the murine proadipocyte stem cell line 3T3-T. By exposure to human plasma, 3T3-T cells can be induced to undergo nonterminal and then terminal differentiation. DNA strand breaks were evaluated using the technique of alkaline elution. No difference was detected among stem, nonterminally differentiated, and terminally differentiated cells in the initial levels of radiation-induced DNA strand breaks. Each of the strand break dose response increased as a linear function of gamma-ray dose. The strand breaks induced by 4 Gy rejoined following biphasic kinetics for each cell type. At each time point examined after irradiation, however, the percentage of strand breaks that had not rejoined in terminally differentiated cells was three to six times greater than in stem cells. The rate of strand break rejoining in nonterminally differentiated cells was of an intermediate value between that of the stem and of the terminally differentiated cells. These results indicate that, at least for 3T3-T cells, differentiated cells have a reduced capacity for DNA repair.

Exposure of certain cell lines to the differentiation-inducing agent N-methylformamide (NMF) enhances their radiosensitivity. As part of an attempt to elucidate the mechanism of NMF-induced radiosensitization, we examined the effects of NMF on chromatin structure, as reflected by changes in DNA-protein cross-links (DPCs) and the chromatin protein/DNA ratio, in two cell lines, clone A and HCA-1. Both lines form a better-differentiated phenotype upon exposure to NMF, yet only clone A is radiosensitized. Ionizing radiation induced DPCs in a linear manner beginning at about 10 Gy and continuing to at least 50 Gy in both cell types. NMF treatment of HCA-1 cells did not affect the background level of DPCs, but it enhanced the formation of radiation-induced DPCs at each dose tested. In clone A cells, NMF exposure elevated the DPC background level more than two-fold, and modified radiation-induced DPCs. The dose response for radiation-induced DPCs in NMF-treated clone A cells consisted of a linear increase up to 12.5 Gy, which was greater than in untreated cells, followed by a plateau level of DPCs out to 50 Gy, the highest dose tested. NMF treatment of clone A, but not HCA-1, cells also increased the chromatin protein/DNA ratio by about 30-35%. In clone A cells, the increases in DPC background level and chromatin protein/DNA ratio as a function of NMF exposure time followed a pattern similar to that of the enhancement of radiosensitivity. These data suggested that modifications of chromatin structure, not involved in differentiation, may be associated with the radiosensitizing actions of NMF.

The effects of the differentiation-inducing polar solvent dimethylsulfoxide (DMSO) on the in vitro response of murine hepatocarcinoma cells to cisplatinum, BCNU, and melphalan were investigated using the sister chromatid exchange (SCE) and cell survival assays. Growth of cells in medium containing 2 per cent DMSO enhanced drug-induced SCEs and cell kill. In order for the enhancement to occur, cells had to be exposed to DMSO for at least 48 h prior to drug treatment. The presence of DMSO during drug treatment did not affect cell response to the three chemotherapeutic agents. The enhancement of chemosensitivity was eliminated within 24 h of DMSO removal. These data suggest that the differentiation-inducing polar solvents may provide antineoplastic benefits when administered in combination with standard chemotherapeutic agents.

The differentiation-inducing agent N-methylformamide (NMF) enhances the sensitivity of some cell lines to ionizing radiation. To elucidate the mechanism of NMF-mediated radiosensitization, we examined the effects of this agent on gamma-ray-induced DNA double-strand breaks and micronuclei in two cell lines, clone A (human colon carcinoma) and HCA-1 (murine hepatocarcinoma). Both cell lines form a better differentiated phenotype upon exposure to NMF, yet only clone A is radiosensitized. The neutral (pH 9.6) elution assay was used to evaluate the effects of this maturational agent on radiation-induced double-strand breaks in these cell lines. Exposure of HCA-1 cells to NMF had no effect on the level of DNA double-strand breaks induced by gamma rays. In clone A cells, however, exposure to NMF enhanced the initial formation of gamma-ray-induced double-strand breaks at each dose tested. The repair of double-strand breaks in both cell lines was not influenced by NMF. As a measure of chromosome fragmentation after irradiation, we evaluated micronuclei using the cytokinesis block method. Exposure to NMF had no effect on radiation-induced micronuclei formation in HCA-1 cells yet significantly enhanced the frequency of micronuclei induced by radiation in clone A cells. In clone A cells, the increases in radiation-induced double-strand breaks and micronuclei as a function of NMF exposure time reached maximums by approximately 72 h. These data suggest that NMF-mediated radiosensitization is the result of an increase in the initial level of radiation-induced DNA double-strand breaks.

Twenty-four hours after a single dose (50 microgram, s.c.) of estradiol benzoate (EB), rat testicular microsomal heme and cytochrome P-450 were decreased to 72 and 76% of control levels respectively. Treatment of rats with human chorionic gonadotropin (hCG) resulted in elevated levels of microsomal heme and cytochrome P-450 and increased activity of delta-aminolevulinic acid (ALA) synthase (EC 2.3.1.37). However, the hCG-mediated elevations of testicular microsomal heme and cytochrome P-450 content failed to occur in animals treated with EB. To investigate the possibility that the observed effect of EB was mediated through the pituitary, studies were conducted with hypophysectomized animals. The increased microsomal heme and cytochrome P-450 content mediated by hCG in hypophysectomized animals was again prevented by administration of EB. The elevated activity of testicular mitochondrial ALA synthase produced by hCG in both intact and hypophysectomized animals was not affected by EB. Incorporation of [14C]ALA into microsomal heme was depressed 60% 12 hr following a single dose of EB (50 microgram, s.c.). These data suggest that EB depresses testicular microsomal heme and cytochrome P-450 content by inhibiting the synthesis of heme at an enzymatic reaction other than ALA synthase.

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.

The effects of the differentiation-inducing agent N-methylformamide (NMF) on the radiation response of ten primary human tumor cell cultures were investigated. Cell survival was determined using an adhesive tumor cell culture system. NMF (1%) was added to cultures on Day 1 and was left for 6 days; cultures were irradiated with graded doses of X rays (1.0-6.0 Gy) on Day 4. Using survival at 2.0 Gy as a comparative endpoint, eight of ten cultures tested exhibited enhanced radiosensitivity upon exposure to NMF. In sensitized cultures, the dose-enhancement factors ranged from 1.3 to 2.5. The NMF-mediated radiosensitization did not appear to be dependent on the histologic cell type. The results presented support previous data obtained from established cell lines and suggest that NMF may offer clinical benefits against a variety of tumor types when used in combination with radiotherapy.

Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) produces hirsutism, alopecia, and chloracne, symptoms that suggest a possible alteration of endocrine function. Therefore, the effects of TCDD on rat testicular cytochrome P-450 content were investigated. Forty-eight hours after a single, oral dose of TCDD (25 microgram/kg) testicular microsomal cytochrome P-450 levels were depressed by approximately 24%. Microsomal cytochrome P-450 continued to decrease to 62% of control levels at 4 days and remained at approximately the same levels 7 days following treatment. Testicular microsomal heme content exhibited a similar pattern after administration of TCDD. No alterations in testicular delta-aminolevulinic acid (ALA) synthase were detected. The incorporation of [14C]ALA into microsomal heme was decreased to approximately 36% of control values at 24 hr after TCDD administration. Testicular weights were not altered during the 7-day experimental period. These data suggest that TCDD depresses cytochrome P-450 levels in the rat testis through an inhibition of the synthesis of testicular heme.

The effect of the polyamine biosynthesis inhibitor alpha-difluoromethylornithine (DFMO) on the in vitro radiation response of Clone A human colon adenocarcinoma cells was investigated. Analysis of intracellular polyamine levels showed that exposure of Clone A cells to 1 mM DFMO for 96 h reduced putrescine and spermidine to nondetectable levels, while spermine was decreased by approximately 50%. This DFMO treatment protocol enhanced the radiosensitivity of Clone A cells, which was reflected by a decrease in both the Do and Dq. The addition of putrescine (1 mM) for the final 48 h of DFMO exposure restored polyamine levels and returned clone A radiosensitivity to that of control cells. These results indicate that polyamine depletion by DFMO sensitizes Clone A tumor cells to ionizing radiation.

In these studies we have used wild-type Chinese hamster ovary cells (AA8) and a mutant cell line (UV-41) deficient in excision repair to compare sister chromatid exchange (SCE) induction after X irradiation under oxic and hypoxic conditions. X irradiation of AA8 cells under oxic conditions induced only a slight increase in SCEs, whereas at each dose tested a significantly greater number of SCEs were induced in hypoxic cells. When AA8 cells were X-irradiated and the addition of bromodeoxyuridine (BrdU) was delayed for 20 h to allow DNA lesions to be repaired, the levels of SCEs detected in both oxic and hypoxic cells returned to background levels. X irradiation of UV-41 cells also induced only a slight increase of SCEs in oxic cells, whereas a significant number of SCEs were induced in hypoxic cells. However, in contrast to results with AA8 cells, when hypoxic UV-41 cells were X-irradiated and the addition of BrdU was delayed for 20 h, the number of SCEs remained significantly above background levels. In combination with previous alkaline elution data, these results are consistent with the possibility that DNA-protein crosslinks are responsible for the SCEs induced by X irradiation of hypoxic cells. Irrespective of the mechanism(s) involved, the data presented suggest that the SCE assay may potentially aid in the detection of hypoxic 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.

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.

The sister chromatid exchange (SCE) assay has been used to predict the chemosensitivity to 1,3-bis(2-chloroethyl)-1-nitrosourea of various neoplastic cell subpopulations in eight cell lines derived from human brain tumors. Because the SCE assay is based on analysis of individual cells, data obtained can be plotted as frequency histograms of SCEs per chromosome, and the range of chemosensitivities of cell subpopulations can be identified easily. Results suggest that the SCE assay has predictive value as a clinical assay for drugs for which a strong correlation between cell kill and induction of SCEs has been established.