Effect of alpha-difluoromethylornithine on L-phenylalanine mustard-induced cytotoxicity and DNA interstrand cross-linking in a human cell line in vitro.

We compared L-phenylalanine mustard (L-PAM)-induced cytotoxicity and DNA cross-linking with and without a 42-h preincubation with the ornithine decarboxylase inhibitor alpha-difluoromethylornithine (DFMO, 1 mM) in a human lymphoma cell line. The combination showed increased toxicity with a Do ratio of 1.6. L-PAM-induced DNA protein cross-linking as measured by alkaline elution was not altered by a DFMO pretreatment. DNA interstrand cross-linking was increased when L-PAM-treated cells were pretreated with DFMO. The differences occurred between 12 and 24 h following the L-PAM treatment. Peak protein cross-linking occurred 6 h following L-PAM removal with or without DFMO pretreatment. While peak interstrand cross-linking occurred 6 h following L-PAM removal, the DFMO-pretreated cells maintained higher cross-link levels longer than did control cells. The increase in interstrand cross-linking seen in DFMO-pretreated cells was maintained at several different L-PAM doses. The increased cytotoxicity could not be accounted for by the increased cross-linking alone. We have postulated that DFMO pretreatment results in a delay in the appearance of a cross-link removal system. The differences seen between results using these human cells and previous reports using rodent cells are discussed.

Use of a highly sensitive assay to analyze the excision repair of dimer and nondimer DNA damages induced in human skin fibroblasts by 254-nm and solar ultraviolet radiation.

The excision repair of nondimer DNA damages induced in normal human skin fibroblasts exposed to the Mylar-filtered UV produced by a fluorescent sunlamp was investigated. This work was accomplished through the development of a modification of the bromodeoxyuridine photolysis assay that greatly increases the sensitivity of this assay. This enhancement in sensitivity was achieved through use of alkaline elution to measure the DNA strand breakage produced by the photolysis of bromodeoxyuridine incorporated into the DNA through excision repair. Using this modified bromodeoxyuridine photolysis assay, it was found that the solar UV-induced nondimer DNA damages appear to have been repaired by a short patch repair mechanism in which a small number of nucleotides (two to four) were inserted into the repaired site. This is in contrast to the long patch repair process involved in the excision of cyclobutane pyrimidine dimers in which approximately 40 nucleotides were inserted into each repaired region.

Cytotoxicity and DNA damage caused by the azoxy metabolites of procarbazine in L1210 tumor cells.

Procarbazine, a chemotherapeutic hydrazine, is thought to be metabolized to an alkylating species similar to methyl carbonium ion by multistep reactions involving cytochrome P-450, monoamine oxidase, and cytosolic enzymes. The DNA-damaging and cytotoxic potential of procarbazine and its metabolites in murine L1210 leukemia tumor cells in vitro was determined using alkaline elution techniques and extrapolation of growth curves. Neither procarbazine nor any of the chemical degradation products (except for the aldehyde derivative at high concentrations) caused significant amounts of DNA strand breakage. The primary enzymatic oxidation product, azo-procarbazine, did not produce strand breakage. However, exposure of the cells to either of the two isomers of azoxy-procarbazine led to significant DNA damage and cytotoxicity. DNA damage included both single-strand breaks and alkali-labile sites. At equimolar concentrations, the azoxy 2 isomer of procarbazine caused 14 to 20 times more DNA damage than did the azoxy 1 metabolite. When cell growth is expressed as percentage survival of L1210 cells, the azoxy 2 isomer was approximately 7-fold more toxic than the azoxy 1 metabolite. The other metabolites tested showed little or no cytotoxicity. L1210 cells were shown to contain little or no cytochrome P-450 or monoamine oxidase activity, which may account for the lack of toxicity of the parent drug or the primary oxidative metabolite, azo-PCZ, to these cells. The conversion of procarbazine to the azoxy-procarbazine isomers in vivo must occur in cells which contain these enzymes, such as liver. However, the azoxy isomers of procarbazine were metabolized in L1210 cells, presumably leading to the DNA or cytotoxic damage observed.

Differential cytotoxicity and DNA cross-linking in normal and transformed human fibroblasts treated with cis-diamminedichloroplatinum(II).

DNA interstrand cross-linking had been found previously to correlate with differences in sensitivity among human cell strains treated with chloroethylnitrosoureas. These differences had been attributed to the presence or absence of a specific DNA repair mechanism. The current work addressed the question of whether another DNA cross-linking agent, cis-diamminedichloroplatinum(II) (cis-Pt), would exhibit analogous differences between cell types. A normal human embryo cell strain (IMR-90) was compared with an SV40-transformed line (VA-13). Interstrand cross-linking and DNA-protein cross-linking were assayed by alkaline elution. As in the case of chloroethylnitrosoureas, the cytotoxicity differences with cis-Pt correlated with differences in interstrand cross-linking. The relative sensitivity of the cell lines to cis-Pt, however, was reversed. Similar DNA-protein cross-linking levels in the two cell lines excluded a difference in cis-Pt uptake or intracellular metabolic drug activation or inactivation prior to DNA interaction. It was concluded that the DNA repair mechanism that prevents interstrand cross-linking by chloroethylnitrosoureas does not prevent interstrand cross-linking by cis-Pt. Interstrand cross-linking by cis-Pt may be prevented by an independent mechanism.

Comparative studies of DNA cross-linking and cytotoxicity in Burkitt's lymphoma cell lines treated with cis-diamminedichloroplatinum(II) and L-phenylalanine mustard.

Three Burkitt's lymphoma cell lines were studied for their response to cis-diamminedichloroplatinum(II) (cis-DDP) and L-phenylalanine mustard (L-PAM) with the objective of relating cytotoxicity to DNA cross-linking in human cells. Cytotoxicity was measured by cell proliferation and colony formation assays. DNA interstrand and DNA-protein cross-linking were measured by alkaline elution. Two of the cell lines showed quantitative agreement between cytotoxicity and DNA cross-linking assays following treatment with cis-DDP or L-PAM. The correlation with DNA-protein cross-linking was not as good as with interstrand cross-linking. With the third cell line, cytotoxicity and interstrand cross-linking were correlated in response to L-PAM but not in response to cis-DDP. When this line was treated with cis-DDP, cross-linking was low, but cytotoxicity was high. This line differed from the other two lines in that, following treatment with either cis-DDP or L-PAM, substantial cell lysis or disruption occurred 12 to 14 hr following treatment. The results are consistent with a general correlation between DNA cross-linking and cytotoxicity in human cells treated with bifunctional agents. An exceptional cell type, however, is described which appears to have a reduced tolerance to certain types of DNA damage, the reduced tolerance being expressed by early cell lysis or disruption.

Surveillance neuroimaging to detect relapse in childhood brain tumors: a Pediatric Oncology Group study.

PURPOSE: To investigate the prognostic significance of surveillance neuroimaging for detection of relapse among children with malignant brain tumors. PATIENTS AND METHODS: A historical cohort study examined all children who experienced relapse from 1985 to 1999 on one of 10 Pediatric Oncology Group trials for malignant glioma, medulloblastoma, or ependymoma. RESULTS: For all 291 patients (median age at diagnosis, 8.2 years), median time to first relapse was 8.8 months (range, 0.6 to 115.6 months). Ninety-nine relapses were radiographic, and 192, clinical; median time to relapse was 15.7 versus 6.6 months, respectively (P = .0001). When stratified by pathology, radiographic and clinical groups showed differences in median time to relapse for malignant glioma (7.8 v 4.3 months, respectively; P = .041) and medulloblastoma (23.6 v 8.9 months, respectively; P = .0006) but not ependymoma (19.5 v 13.3 months, respectively; P = .19). When stratified by early (< 8.8 months) or late (> or = 8.8 months) time to relapse, 115 early relapses were clinical, and 32, radiographic; for late relapses, 77 were clinical, and 67, radiographic (P = .001). Overall survival (OS) from relapse was significantly longer for radiographic compared with clinical detection (median, 10.8 months; 1-year OS, 46% v median, 5.5 months; 1-year OS, 33%; P = .002), but this trend did not retain significance when analyzed by pathology subgroups. CONCLUSION: Surveillance neuroimaging detects a proportion of asymptomatic relapses, particularly late relapses, and may provide lead time for other therapies on investigational trials. During the first year after diagnosis, radiographic detection of asymptomatic relapse was infrequent. A prospective study is needed to formulate a rational surveillance schedule based on the biologic behavior of these tumors.

Relationship between life expectancy and endogenous DNA single-strand breakage, strand break induction and DNA repair capacity in the adult housefly, Musca domestica.

The objective of this study was to investigate the relationship between genomic damage and the physiological rate of aging. Endogenous DNA single-strand breaks, susceptibility of DNA to exogenously induced strand breaks and the capacity to repair strand breakage were compared, using the alkaline elution technique, in flies of the same chronological age but with different life expectancy. Distinctions between physiological and chronological ages were made (1) by experimentally altering the life spans of houseflies by varying the level of physical activity, and (2) by phenotypic selection of short- and long-lived cohorts from the same population. The degree of endogenous DNA single-strand breaks was found to be unrelated to physiological age. However, flies selected for relatively shorter life expectancy exhibited a greater susceptibility to exogenously-induced (gamma-irradiation) single-strand breakage. Flies with a longer life expectancy exhibited a more efficient repair capacity to reverse single-strand breakage than those with a shorter life expectancy.

Regression on oxymetholone-induced hepatic tumors after bone marrow transplantation in aplastic anemia.

Treatment of acquired aplastic anemia with androgens has been occasionally associated with the development of hepatic tumors. We have studied a 13-year-old boy with idiopathic aplastic anemia in whom oxymetholone treatment was associated with a partial hematological remission. Thirty-four months later, however, the patient developed multiple hepatic tumors. When oxymetholone therapy was discontinued, the aplastic anemia relapsed. He then underwent bone marrow transplantation from his HLA-A, B, and D-compatible sibling. This was followed by hematological and immunological reconstitution. The hepatic tumors underwent progressive regression after bone marrow transplantation. The patient is now 3 years post-bone marrow transplantation and is in complete remission of his aplastic anemia with no evidence of detectable liver tumors.

alpha-Difluoromethylornithine effects on nitrosourea-induced cytotoxicity and crosslinking in a methylation excision repair positive (MER+) human cell line.

We investigated the cytotoxic effects of nitrosoureas with and without a 42-hr preincubation with the ornithine decarboxylase (EC 4.1.1.17) inhibitor alpha-difluoromethylornithine (DFMO, 1 mM) in a MER+ (methylation excision repair positive) human cell line. DFMO combined with a chloroethyl nitrosourea [1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) or 1-(2-chloroethyl)-1-nitrosourea (CNU)] yielded increased toxicity with D37 ratios of 1.9 and 3.3 respectively. There was no enhanced toxicity with the monofunctional nitrosourea 1-ethyl-1-nitrosourea (ENU). BCNU or CNU did not induce DNA-DNA interstrand crosslinks in cells with or without a DFMO pretreatment. DNA single-strand breakage was not increased by addition of DFMO. BCNU-induced DNA-protein crosslinking was decreased in cells pretreated with DFMO. These findings are similar to those in MER- cells in that the chloroethyl carbonium alkylating species is required for the enhanced cytotoxicity seen with DFMO. The ability to form DNA interstrand crosslinks, however, does not appear to be necessary for this toxicity enhancement.

cis-Diamminedichloroplatinum(II) (DDP)-induced crosslinking and crosslink removal in L1210 cells in vitro after theophylline co-treatment.

The present study investigated the mechanism by which theophylline decreases cis-diamminedichloroplatinum(II) (DDP)-induced DNA crosslinking in L1210 cells. Alkaline elution of DNA from L1210 cells treated with DDP in the presence and absence of 1 mM theophylline showed that theophylline decreased interstrand crosslinking by 20%. DNA-protein crosslinking (PXL) immediately following platinum removal (used as a measure of active drug delivered to the cell nucleus) was not altered by theophylline nor were the kinetics of either DNA interstrand (ISC) or DNA-protein crosslink formation after DDP treatment changed by the presence of theophylline. Peak protein crosslinking occurred 6 hr and peak interstrand crosslinking occurred 12 hr after DDP removal. We measured crosslink removal by using thiourea to block the conversion of platinum monoadducts to crosslinks. DNA-protein crosslinks were removed more rapidly in the presence of theophylline. There was no change in interstrand crosslink removal rate when theophylline was present. We conclude that the addition of theophylline to DDP treatment results in decreased amounts of DNA interstrand crosslinking most likely by increasing the removal of DPP-DNA monoadducts. This latter process may be reflected by the increased removal rates for DNA-protein crosslinks.

Analysis of the excision repair of nondimer DNA damage induced by solar ultraviolet radiation in ICR 2A frog cells.

The excision repair of solar uv-induced nondimer DNA damage was examined in ICR 2A frog cells through the use of the bromodeoxyuridine (BrdUrd) photolysis assay. A relatively pure population of nondimer DNA photoproducts was induced by irradiation of ICR 2A cells with the Mylar-filtered solar ultraviolet (uv) wavelengths produced by a fluorescent sunlamp followed by exposure to photoreactivating light (PRL) which removes most of the small yield of pyrimidine dimers induced by this treatment. Cultures of cells were also exposed to 254 nm uv, which induces primarily dimers, and 60Co gamma rays. Through use of a modification of the BrdUrd photolysis assay possessing enhanced sensitivity, it was found that the solar uv-induced nondimer DNA damage was repaired by a short patch repair mechanism in which less than approximately 20 nucleotides are inserted into a repaired region. Similar results were also obtained for gamma-irradiated cells. In contrast, excision repair of 254-nm-induced dimers was accomplished by a long-patch process in which an average of about 180 nucleotides are inserted into the repaired sites.

EDTA alkaline elution characteristics and measurement of DNA damage in unlabeled DNA using Hoechst 33258 fluorescence.

Hoechst 33258 fluorescence of single stranded DNA has been used to perform alkaline elution with unlabeled DNA. The high background fluorescence of "standard" elution solutions has prompted others to use EDTA but the elution characteristics of DNA in EDTA-containing solutions and the comparability of results with those using "standard" tetrapropyl ammonium hydroxide solutions have not previously been examined. We report here the elution characteristics of DNA in EDTA and the relevant parameters for the successful use of EDTA as an elution solution. An increase in elution pH to 12.4 is required but elution solutions of higher pH cause alkaline hydrolysis of undamaged DNA. Drug-treated DNA from which DNA-protein crosslinks have been removed can be completely removed from the filters at the end of the elution by a Pronase filter digestion. The simplest and most efficient removal of DNA-protein crosslinks is through the inclusion of proteinase-K in an SDS containing lysis solution. EDTA elution can measure interstrand crosslinks and single strand breaks as easily as is performed using radiolabeled DNA under "standard" elution conditions and requires only 1.5-2 x 10(6) cells per elution filter. DNA-protein crosslinking measurements were unsatisfactory, however, since even the Pronase digestion failed to completely remove protein-crosslinked DNA from the elution filters.

DNA-protein crosslinking in normal human skin fibroblasts exposed to solar ultraviolet wavelengths.

Three normal human skin fibroblast cell lines were exposed to the simulated solar UV radiation produced by a fluorescent sunlamp under conditions in which the wavelength components shorter than either 295, 305 or 315 nm were excluded. The level of DNA-protein crosslinks (DPC) was then measured in those cells using the alkaline elution technique either immediately after irradiation or following a 24 h incubation. In each case, cells were exposed to fluences that induce similar levels of DPC. For cells exposed to 10 kJ m(-2) of sunlamp UV > 295 nm, the level of DPC exhibited a 2-5-fold increase following incubation. In contrast, 40-100% of the DPC were removed upon incubation of cells irradiated with either 100 kJ m(-2) of sunlamp UV > 305 nm or 150 kJ m(-2) of sunlamp UV > 315 nm. A major difference between the effects induced by these wavelength regions is that, in addition to DPC, a very high level of pyrimidine dimers is also produced by sunlamp UV > 295 nm, whereas much lower dimer yields result from treatment with either sunlamp UV > 305 nm or sunlamp UV > 315 nm. A potential role for type II DNA topoisomerase in the formation of these DPC resulting from either the change in conformational structure caused by the presence of a high level of dimers or an involvement of this enzyme in dimer excision repair is discussed.

Theophylline does not reverse DNA replicon initiation inhibition in human cells resistant to alkylating agent-theophylline killing synergism.

The human Burkitt's lymphoma cell line BHM fails to show synergistic killing by alkylating agents in the presence of theophylline. Nitrosoureas (BCNU and CNU), a mustard agent (L-phenylalanine mustard), and a platinum coordination complex (cis-diamminedichloroplatinum-II) did not show increased cytotoxicity when cells were treated in the presence of theophylline. Despite varying abilities of the drugs to induce DNA damage in BHM cells (no DNA interstrand cross-linking with nitrosoureas or platinum and significant interstrand cross-linking following L-PAM treatment) theophylline did not alter the pattern of DNA damage. DNA interstrand cross-linking following treatment by L-PAM with theophylline was slightly decreased from that seen with L-PAM alone. All three drugs induced DNA replicon initiation inhibition in BHM cells as measured both by alkaline sucrose gradient sedimentation and pH step alkaline elution. As opposed to cell lines where methylxanthines increase alkylating agent cytotoxicity, theophylline and caffeine failed to reverse the drug-induced replicon initiation inhibition seen in BHM cells. These findings support the hypothesis that the synergistic killing seen in some cell lines with alkylating agents and methylxanthines is due to the reversal of replicon initiation inhibition by the methylxanthines.

Theophylline reversal of alkylator-induced replicon initiation inhibition: no correlation with DDP-induced cytotoxicity.

The synergistic cytotoxic activity exhibited by bifunctional alkylating agents in the presence of methylxanthines has been associated with methylxanthine-induced reversal of alkylator-induced DNA replicon initiation inhibition. This has also been seen with methylxanthines and ionizing irradiation. Methylxanthines do not appear exacerbate drug or ionizing radiation-induced damage. We report here a situation in which methylxanthine-induced reversal of DNA replicon initiation inhibition is not associated with increased cytotoxicity of the alkylator. Murine L1210 leukemia cells were assayed for cytotoxicity following treatment with either L-PAM or cis-DDP in the presence or absence of theophylline. Theophylline increased the cytotoxicity seen after L-PAM treatment but failed to increase the cis-DDP induced cytotoxicity. Analysis of pulse-labeled DNA on alkaline sucrose gradients revealed the expected decrease in DNA replicon initiation in L1210 cells treated with either L-PAM or cis-DDP. Theophylline had no effect on replicon initiation in untreated cells. Theophylline reversed the replicon initiation inhibition in cells treated with either L-PAM or cis-DDP. The reason for the apparent lack of added toxicity of the replicon initiation inhibition reversal in L1210 cells treated with theophylline and DDP is unknown.

Theophylline release of replicon initiation inhibition by nitrosoureas correlates with the synergistic killing in L1210 leukemia in vitro.

This report demonstrates the synergistic killing of murine L1210 leukemia in vitro by BCNU and theophylline as has previously been reported in vivo. Synergism is also seen with the related nitrosourea CNU plus theophylline. As measured on alkaline sucrose gradients and by pH-step alkaline elution, the nitrosourea-induced inhibition of DNA replicon initiation is completely reversed in the presence of theophylline. DNA interstrand crosslinking, the damage which usually correlates with nitrosourea cytotoxicity, is not increased by the combination of nitrosourea plus theophylline. At high nitrosourea doses, this interstrand crosslinking is reduced in the presence of theophylline. At least part of the mechanism of the two-drug synergism is the theophylline release of nitrosourea-induced DNA initiation inhibition. Some of the results have been presented at the Annual Meeting of the American Association for Cancer Research.

Effect of age on endogenous DNA single-strand breakage, strand break induction and repair in the adult housefly, Musca domestica.

It has been suggested that genomic alterations involving DNA damage and the ability to repair such damage play an important role in cellular senescence. In this study, endogenous DNA single-strand breaks, the susceptibility of DNA to induced strand breakage and the capacity to repair these breaks were compared in postmitotic cells from young (3-day-old) and old (23-day-old) houseflies. DNA single-strand breaks did not accumulate during normal aging in the housefly. However, cells of the old flies exhibited a greater sensitivity to single-strand breakage induced by gamma-radiation and UV light. The capacity to repair these exogenously induced single-strand breaks declined with age. Results do not support the view that DNA single-strand breaks are a causal factor in aging in the housefly. An age-related increase in the susceptibility to undergo single-strand breakage suggests alterations in chromatin during the aging process.

The mechanism of bilirubin-photosensitized DNA strand breakage in human cells exposed to phototherapy light.

Exposure of normal human fibroblasts to visible light (420-490 nm) in the presence of exogenously added 1-100 micrograms/ml bilirubin enhanced the level of DNA strand breakage compared with cells irradiated in the absence of added bilirubin. Treatment of cells in the dark with an irradiated bilirubin solution also induced DNA strand breaks. However, strand breakage was not detected in cells treated with an irradiated bilirubin solution that had been incubated with catalase (H2O2: H2O2 oxidoreductase EC 1.11.1.6). Examination of irradiated bilirubin solutions demonstrated the presence of hydrogen peroxide although, apparently, not at concentrations sufficient to account for the level of DNA strand breakage detected. Hence, irradiation of bilirubin results in the generation of hydrogen peroxide and possibly other peroxides that can cause DNA damage.

DNA-protein crosslinking in normal and solar UV-sensitive ICR 2A frog cell lines exposed to solar UV-radiation.

DNA-protein crosslinks (DPC) were measured following exposure to the solar UV wavelengths produced by a fluorescent sunlamp in ICR 2A frog cells and two solar UV-sensitive mutants derived from this cell line. Approx. 5-7 DPC per 10(10) dalton were induced in these cells by either 150 kJ/m2 of sunlamp UV greater than 315 nm plus photoreactivating light (PRL) or 10 kJ/m2 of sunlamp UV greater than 295 nm. The irradiated cells were then incubated for 0-24 h and the level of DPC measured using alkaline elution. It was found for the ICR 2A cells exposed to sunlamp UV greater than 315 nm that the level of DPC increased about 3-fold during a 2-h postirradiation incubation and then decreased. The mutant cell lines also showed an enhancement in the level of DPC following irradiation, although it was much less pronounced and the levels decreased much more rapidly. In a similar fashion, the level of DPC increased in ICR 2A cells exposed to sunlamp UV greater than 295 nm with more than a 5-fold enhancement after a 4-h incubation. Once again, the mutant cell lines showed an increase in the level of DPC that was smaller and more transient than the effect in the ICR 2A cells. These results suggests that this enhancement in DPC may be indicative of a process that plays a role in cellular survival following solar UV-irradiation.

Hydrogen peroxide induces DNA single strand breaks in respiratory epithelial cells.

The respiratory epithelium is often exposed to oxidant gases, including ozone from photochemical smog and toxic oxygen metabolites released from neutrophils recruited in conditions of airway inflammation. We evaluated DNA single strand break formation by alkaline elution as a measure of oxidant-induced DNA damage to bronchial epithelial cells. Human AdenoSV-40-transformed bronchial epithelial cells (BEAS), subclone R1.4 or nonhuman primate bronchial epithelial cells were cultured in growth factor supplemented Ham's F12 medium on polycarbonate filters. DNA was labeled by incubation with [3H]thymidine. Cells were incubated for 1 h in HBSS or HBSS and increasing concentrations of hydrogen peroxide (H2O2). Cells incubated in H2O2 demonstrated dose-dependent increases in strand break formation, and BEAS cells were more sensitive to H2O2-induced injury than primary bronchial epithelial cells. The addition of catalase or preincubation of cells with the iron chelator desferoxamine prevented H2O2-induced strand breakage. DNA strand break formation may be an important mechanism of oxidant injury in respiratory epithelial cells.