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Normal human skin fibroblasts were exposed to either 0-5 J/m2 of 254-nm ultraviolet (UV) radiation or 0-50 kJ/m2 of the Mylar-filtered UV (greater than 310 nm) produced by a fluorescent sunlamp. These cells were then incubated for 0-20 min in medium containing 10 mM hydroxyurea (HU) and 0.1 mM 1-beta-D-arabinofuranosyl cytosine (ara C), and the yield of DNA strand breaks was measured by means of the alkaline elution technique. For cells irradiated with 254-nm UV, which results primarily in the formation of cyclobutane pyrimidine dimers, a rapid increase in DNA strand breaks was detected following incubation with these metabolic inhibitors. In contrast, only a low level of strand breaks formed in cells incubated with HU and ara C after irradiation with approximately equitoxic fluences of sunlamp UV greater than 310 nm, which mainly causes the induction of nondimer DNA lesions. Hence, these results are consistent with the conclusion that the pathways involved in the repair of nondimer DNA damages induced by UV wavelengths greater than 310 nm differ from the repair of pyrimidine dimers.

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.

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.

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.

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.

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.

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.

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.

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.

Serial studies of human leukemic lymphoblasts (CCRF-CEM line) cultured with 0.25 to 2.5 microM VP-16-213 for 0 to 6 hr indicated that the mechanism of cytotoxicity of this compound involves a primary effect on DNA. The most striking early change shown by flow cytometry in VP-16-213-treated cells was a delay in S-phase transit before arrest of cells in G2. Coinciding with this S-phase delay was a selective inhibition of thymidine incorporation into DNA as well as concentration-dependent scission of DNA strands. Using alkaline elution methods, we were able to detect DNA breakage at concentrations of VP-16-213 well below the level required to demonstrate kinetic effects or inhibition of DNA synthesis. These data suggest that DNA strand scission is the initial event in the sequence of kinetic and biosynthetic changes leading to growth inhibition and death of VP-16-213-treated cells. Inhibition of replicon initiation due to strand scission is a plausible explanation for the cytotoxic action of this podophyllotoxin derivative.

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.

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.

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.

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.

Normal human fibroblasts were exposed to light from three types of lamps commonly used for phototherapy treatment of neonatal hyperbilirubinemia. These irradiations were performed in either the presence or absence of exogenously added bilirubin. Using the alkaline elution assay, we found that each phototherapy lamp induced strand breaks in the DNA of exposed cells. However, the cross sections for DNA strand breakage were increased 30-40-fold when cells were irradiated in the presence of 100 micrograms/ml bilirubin. Hence, bilirubin acts as a photosensitizing agent enhancing the level of DNA damage in cells exposed to phototherapy light.