Katherine A. Kennedy

Concurrent unilateral chromatid damage and DNA strand breakage in response to 6-thioguanine treatment

The delayed cytotoxicity of 6-thioguanine (TG) may relate to the arrest of cells in G2 upon completion of one cell cycle after drug exposure. In Chinese hamster ovary (CHO) cells, both the unilateral chromatid damage in G2 chromosomes, determined by induction of premature condensed chromosome condensation [Maybaum and Mandel, Cancer Res. 43, 3852 (1983)], and incorporation of TG into DNA resulting in DNA strand breakage [Christie et al., Cancer Res. 44, 3665 (1984)] were correlated with cytotoxicity. We have studied the correlation between strand breakage and unilateral chromatid damage in L1210 cells. DNA breaks were detected only when cells were treated with TG (0.25 microM) for one cell cycle time (12 hr) followed by 12 hr in drug-free medium containing [3H]thymidine (TdR) to label the DNA. After simultaneous incubation of cells with drug and label during the first or second 12-hr period, strand breaks were not found. Strand breaks increased with dose, which correlated with greater cytotoxicity (0.01 to 0.25 microM). Treatment of cells with 0.25 microM TG for 12 hr, and transfer to drug-free medium for 12 hr prior to making prematurely condensed chromosomes (PCC), resulted in unilateral chromatid damage. Prominent curving of G2 chromosomes with gapping and diffuse staining of one of the sister chromatids occurred. The 4-fold increase in the percentage of cells in G2 compared with control cells suggested G2 arrest. When cells were treated with TG for 12 hr and PCC made immediately, neither the arrest of cells in G2 nor unilateral chromatid damage was observed. These data suggest that strand breaks and unilateral chromatid damage occur in the second cell cycle after TG exposure and that this damage may be important in TG-delayed cytotoxicity.

Mitomycin-C as a prototype bioreductive alkylating agent: In vitro studies of metabolism and cytotoxicity

The bioreductive alkylating agents are prodrugs for chemotherapy which are enzymatically reduced within cells to species capable of alkylating biological molecules and producing cytotoxic damage. Studies presented in this report show that MIT-C has the characteristics expected of a bioreductive alkylating agent: activation to alkylating species occurs more readily under hypoxic conditions and the drug is selectively cytotoxic to hypoxic cells.

Cellular pharmacology of quinone bioreductive alkylating agents

The cellular pharmacology of the mitomycin bioreductive alkylating agents is complex. This reflects in part the chemical characteristics of these quinones, which have multiple sites of reactivity and the capacity to produce a large number of different lesions of biological importance. Moreover, at least six different enzymes are capable of activating these compounds; the nature of the active species and the resultant biological lesions can vary with the activating enzyme. The relative activities of these reductases vary in different cell lines and can be modulated by pH and oxygenation. The effects of a quinone bioreductive alkylating agent therefore depend upon both the cell line and the microenvironment. DNA damage appears to be critical to the cytotoxic effects of these compounds. Both monoadducts and bis-adducts (forming interstrand and intrastrand crosslinks) have been identified in DNA from drug-treated cells. The pattern of adduct formation varies with the compound and the environment. Alkaline elution studies suggest a correlation between DNA cross-linking and cytotoxicity, both in air and in hypoxia. The rate of production of oxygen radicals and the importance of radical reactions in producing cytotoxic damage vary for different quinones and for different environments. While the potency of the bioreductive quinones varies with their redox potential, the direction and magnitude of the oxic/hypoxic differential cannot yet be predicted from the structures.

Chemical modulation of bleomycin induced toxicity

Both lidocaine (LIDO) and the calmodulin (CaM) antagonists, pimozide (PIM) and trifluoperazine (TFP), enhanced bleomycin (BLM) induced cytotoxicity and DNA damage. The toxicity with the combination of BLM and CaM antagonists represented true pharmacological synergism and was observed with the addition of the CaM antagonist either during or after BLM exposure. Additionally, the DNA damage of BLM and the BLM-like drugs, talisomycin S10b (TAL) or peplomycin (PEPLO), was also enhanced by CaM antagonists. LIDO, which similarly increased the lethal effects and DNA damage of BLM in L1210 cells, was also effective only during or after BLM exposure. The data presented here indicate that the modulation of toxicity seen with these drug combinations is reflected by changes in DNA integrity. Furthermore, these data suggest that the inhibition of DNA repair processes may be at least partially responsible for the enhanced toxicity and DNA damage when CaM antagonists or LIDO are added to BLM.

Involvement of the cellular vacuolar system with the cytotoxicity of bleomycin-like agents.

The role of acidic cellular organelles in regulating the toxicity of selected antitumor drugs was studied with L1210 cells using modifiers of vesicular pH or function. A 1 hr exposure to a non-toxic concentration of the acidotropic weak base ammonium chloride increased the lethality of bleomycin A2 (BLM A2), demethyl BLM A2, peplomycin, and talisomycin S10b to L1210 cells grown in culture. Enhanced BLM lethality was also seen with the lysosomal disruptive agents verapamil and diltiazem. The increased lethality with verapamil and BLM A2 was schedule dependent, being seen only when cells were exposed to the drugs simultaneously or to BLM A2 first and then verapamil. Non-toxic concentrations of the monovalent cationic ionophore monensin also increased the cytotoxicity of BLM A2 and talisomycin S10b but not doxorubicin. This enhanced cytotoxicity seen with monensin occurred without an increase in either cell associated BLM A2 or single-strand DNA damage as measured by alkaline elution. We propose that acidic cellular organelles or their contents participate in controlling the cytotoxicity of the BLM class of antitumor agents.

Porfiromycin as an adjunct to radiotherapy in young and old mice

Radiobiological data and measurements with O2 microelectrodes show that EMT6 tumors implanted into aged mice have a higher proportion of radioresistant, hypoxic cells than do tumors implanted into young adult animals; radiation is less effective in killing cells in tumors in old mice than in tumors in young adult mice. The studies reported here examine the effects of porfiromycin (POR), a bioreductive alkylating agent shown previously to be preferentially toxic to hypoxic EMT6 cells in vitro and in solid tumors in young adult mice. POR was effective in attacking the hypoxic cells of tumors in aged mice; regimens combining POR with x-rays overcame the radioresistance of tumors in the old animals. Comparisons of the distribution of 3H-labeled POR in young and old mice showed that tumors in aged mice had a slightly larger proportion of areas with necrotic features, which bound higher levels of tritiated POR than did healthy tumor regions without necrotic features. Studies of histology, lissamine green distributions, binding of tritiated POR, and radiation and POR cytotoxicity suggested that tumors in old mice contained a larger proportion of poorly perfused tumor cells, and that cells in these regions were resistant to radiation and sensitive to POR. Studies of the distribution of POR in normal tissues and of the toxicity of POR to bone marrow progenitor cells (CFU-GM) revealed no differences between young and old animals, showing that the differences observed in tumors reflected differences in the microenvironments within the tumors, rather than differences in the processing of drug in young and old animals.

Effects of Fusarium moniliforme culture extracts and fumonisin B1 on DNA, RNA and protein synthesis by baby hamster kidney cells

Baby hamster kidney cells (BHK-21) were exposed to culture filtrates of 4 Fusarium moniliforme isolates containing varying levels of fumonisin B1 (FMB1) and the effects upon RNA, DNA and protein synthesis were monitored. Cells were also grown on medium amended with FMB1 only for comparison. After 24 h incubation FMB1 (100 μg/100 ml medium) reduced protein synthesis by 4% and by 18% after 48 h. Culture filtrates containing the highest levels of FMB1 also caused the greatest inhibition in protein synthesis after 24 h but after 48 h protein synthesis levels were the same as controls even though the FMB1 level was 360 μg/100 ml. Only FMB1 reduced DNA synthesis, by 8% after 24 h but after 48 h DNA levels had increased by 40 % over controls. The culture filtrates containing the highest levels of FMB1 (360 μg/100 ml) reduced DNA synthesis more than 50% after 24 h and 48 h. Culture filtrates containing lesser amounts of FMB1 in some instances stimulated DNA synthesis and inhibited it in others. There was also no correlation in the level of FMB1 with the inhibition of RNA synthesis by BHK cells. It appears that metabolites other than fumonisin produced by F. moniliforme in culture can affect and both stimulate and inhibit RNA, DNA and protein synthesis by BHK cells.