Donna Kerrigan

Sequential administration of camptothecin and etoposide circumvents the antagonistic cytotoxicity of simultaneous drug administration in slowly growing human colon carcinoma HT-29 cells

We compared the cytotoxicity of simultaneous and sequential combination chemotherapy with camptothecin and etoposide, in slowly growing human colon carcinoma, HT-29 cells. Simultaneous treatments of HT-29 cells with etoposide and camptothecin produced no marked enhancement of cytotoxicity over single agent administration. This finding demonstrates antagonism of one drugs cytotoxicity over the other. When these studies were repeated in sequential treatment protocols, we observed that antagonism could be circumvented if the period between individual drug administration was separated by 6-8 h. The cytotoxicity that was observed with this approach was never more than additive and the order of camptothecin or etoposide administration did not significantly affect the extent of combined cytotoxicity observed. The protective effect of simultaneous camptothecin and etoposide exposure was not due to reduced formation or alterations in the rate of cleavable complex reversal, and protection persisted for a considerably longer period of time than DNA strand breaks. Protection correlated with the kinetics of DNA and RNA synthesis inhibition produced by either drug. Remarkably, full cytotoxic protection could be afforded by one drug over the other, in the presence of only partial inhibition of DNA or RNA synthesis (50-60%). Our findings suggest that sequential rather than simultaneous administration of topoisomerase I and II inhibitors in future cancer chemotherapy schedules will enhance cytotoxicity over single-agent administration.

Cell death induced by topoisomerase inhibitors: Role of calcium in mammalian cells

Although the stabilization of topoisomerase II cleavable complexes by etoposide (VP-16) has been recognized to be important for cell killing, the lethal events following the formation of cleavable complexes remain to be elucidated. In an attempt to characterize the biochemical requirements for VP-16-induced cytotoxicity, we examined the effects of calcium depletion in Chinese hamster DC3F cells. Four-hour preincubation in calcium-free medium or in complete medium containing 5 mM [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) protected against the cytotoxicity of VP-16. Under these same conditions, the VP-16-induced DNA single-strand break frequency in calcium-depleted cells remained similar to that of control cells. Cell-cycle analysis and thymidine pulse incorporation indicated that calcium depletion did not alter DNA synthesis and cell cycle distribution. Drug-induced cytotoxicity was restored progressively within 4-8 hr after calcium-depleted cells were refed with calcium-containing medium. Calcium depletion also protected against the cytotoxicity of camptothecin, hyperthermia and, to a lesser extent, nitrogen mustard and gamma radiation in DC3F cells. Similar results were obtained in human colon carcinoma HT-29 cells. Our results suggest that topoisomerase II-mediated DNA breaks are only potentially lethal and that calcium-dependent cellular processes are required for the cytotoxicity of topoisomerase inhibitors.

Inhibitors of poly-(adenosine diphosphoribose) synthesis slow the resealing rate of x-ray induced DNA strand breaks

The rate of resealing of x-ray-induced DNA single-strand breaks was measured in L1210 cells in the absence and presence of inhibitors of poly-(adenosine diphosphoribose) synthesis by the alkaline elution technique. Both 5-methylnicotinamide and 3-aminobenzamide slowed, but did not prevent x-ray-induced DNA break resealing. The ability of cellular DNA to repair over 90% of x-ray-induced strand breaks under conditions of poly-(adenosine diphosphoribose) synthesis inhibition suggests that x-ray break resealing may proceed in two ways, one associated with poly-(adenosine diphosphoribose) synthesis and one independent of its synthesis.

Protein-associated deoxyribonucleic acid strand breaks produced in mouse leukemia L1210 cells by ellipticine and 2-methyl-9-hydroxyellipticinium

DNA intercalating agents, including ellipticine, had been found previously to produce protein-associated DNA single-strand breaks and double-strand breaks in mammalian cells. The relationship between these effects on DNA and cytotoxicity could not be determined reliably for ellipticine, because of the poor solubility characteristics of this compound. Studies were therefore carried out using the cationic derivative, 2-methyl-9-hydroxyellipticinium (2-Me-9-OH-E+), which has adequate water solubility and retains antitumor activity. DNA single-strand breaks (SSB) and DNA-protein crosslinks (DPC) were measured using the alkaline elution (pH 12) technique, and double-strand breaks (DSB) were measured by the neutral elution (pH 10) method. The effects of ellipticine and 2-Me-9-OH-E+ were compared in mouse leukemia L1210 cells. Like ellipticine, moderate concentrations of 2-Me-9-OH-E+ produced protein-associated SSB, indicated by the appearance of SSB and DPC at approximately equal frequencies and localized with respect to each other. Below 20 μM (1-hr treatments), the effects of the two drugs were comparable in magnitude. At higher concentrations, ellipticine produced extensive DNA breakage not associated with protein; this is perhaps secondary to an action on membranes or other non-DNA targets. However, 2-Me-9-OH-E+ produced only protein-associated strand breaks, even at 4-fold higher concentrations. The two compounds produced similar and relatively large extents of double-strand scission. The measured DSB/SSB ratio was higher than that produced by X-ray or certain other intercalators that have been similarly studied. The DNA effects of 2-Me-9-OH-E+, unlike those of ellipticine, were readily reversible and, therefore, permitted a meaningful correspondence between the magnitudes of the DNA effects and the inhibition of colony-forming ability. Comparison with two other types of intercalating agents indicated that neither the SSB nor the DSB predicts the magnitude of cell killing.

The reconstitution of higher-order DNA structure after X-irradiation of mammalian cells.

X-ray-induced DNA repair in mouse leukemia (L1210) cells was studied by alkaline elution, which measures the amount of DNA strand breakage, coupled with nucleoid sedimentation, which measures DNA compactness. Two phases of X-ray repair were detected. An initial phase was rapid (t1/2 less than 10 min). During this phase most strand breaks were rejoined and some compaction occurred. After a lag of 1-2 hours, a second phase occurred which exhibited very little or no additional ligation but further compaction of the nucleoid DNA. Both the DNA strand rejoining and initial nucleoid compaction of the first phase were inhibited by 3-ABA2 but not by novobiocin, and the second phase was inhibited by novobiocin but not by 3-ABA. The two phases of reconstitution of nucleoid compactness following X-irradiation are thus distinguishable by their time of occurrence and by their sensitivity to inhibitors of DNA-related enzymes. A coordinated process of ligation followed by compaction may be intrinsic to DNA repair following X-irradiation.

The formation and resealing of intercalator-induced dna strand breaks in isolated l1210 cell nuclei.

Abstract DNA single-strand breaks and DNA-protein crosslinks induced by intercalating agents were measured in mouse leukemia L1210 cell nuclei by the alkaline elution technique. m-AMSA and 5-iminodaunorubicin produced protein-associated DNA breaks but at a lower level than that produced in whole cells. The frequency of drug-induced DNA single-strand breaks and DNA-protein crosslinks were approximately equal. The rapid formation and resealing of DNA breaks produced by m-AMSA were similar to that seen in whole cells. The DNA-protein crosslinks were also reversible after m-AMSA removal. The process by which reversible, protein-associated DNA strand breaks are produced by intercalating agents can now be studied in an isolated chemical system possessing the breaking-rejoining capacity of whole cells.

Ataxia-telangiectasia cells are not uniformly deficient in poly(ADP-ribose) synthesis following X-irradiation

The synthesis of poly(adenosine diphosphoribose) [poly(ADR-R)] follows the DNA strand breakage produced by a number of physical and chemical agents, including X-radiation, and may be important for repair of several types of DNA damage. The reduction or abolition of its synthesis following X-irradiation might explain the enhanced sensitivity of ataxia-telangiectasia (A-T) cells to X-ray. We have examined 8 lines of human fibroblasts (including 4 A-T lines) for stimulation of the synthesis of poly(ADP-R) by X-irradiation. Similar amounts of X-ray-stimulated synthesis of poly(ADP-R) were detected in 4 lines of A-T fibroblasts, and in fibroblasts from a xeroderma pigmentosum (XP) patient, a Fanconis anemia (FA) patient and 2 normal patients. 6 lines of human lymphoblastoid lines were also examined for X-ray-stimulated poly(ADP-R) synthesis. 4 A-T lines displayed an unusually high synthesis of poly(ADP-R) in unirradiated cells compared with 2 normal lines. Despite this complication, some but not all, of the A-T lymphoblastoid lines did synthesize poly(ADP-R) following X-irradiation similarly to the normal lines. Thus, deficient poly(ADP-R) synthesis following X-irradiation is not likely to explain the enhanced sensitivity of all A-T cells to this DNA-breaking agent.

10,11-Methylenedioxycamptothecin, a Topoisomerase I Inhibitor of Increased Potency: DNA Damage and Correlation to Cytotoxicity in Human Colon Carcinoma (HT-29) Cells

We had previously shown that 10,11-methylenedioxy-20-(RS)-camptothecin (MDO-CPT) is a more potent inhibitor of purified DNA topoisomerase I than 20-(S)-camptothecin (CPT). The current studies compared the cytotoxicity and DNA damage induced by MDO-CPT and CPT in the human colon carcinoma cell line, HT-29. MDO-CPT was 7- to 10-fold more potent than CPT both for cytotoxicity (ID50 = 25 vs. 180 nM) and production of DNA single-strand breaks (SSB). Kinetics of SSB formation and reversal were similar for MDO-CPT and CPT. DNA-protein crosslinks (DPC) were also produced by both drugs with a SSB/DPC ratio of 1/1. Moreover, no SSB were detected under non-deproteinizing conditions, indicating that both CPT and MDO-CPT produced protein-linked DNA single-strand breaks. A good correlation between cytotoxic potency and protein-linked DNA single-strand break production was observed for CPT and MDO-CPT, implying a causal relationship between drug-induced cytotoxicity and topoisomerase I inhibition. The sensitivity of human colon HT-29 cancer cells to camptothecins may be a selective phenomenon since these cells normally express natural resistance to current chemotherapeutic drugs, including topoisomerase II inhibitors.

Comparison of DNA scission and cytotoxicity produced by Adriamycin and 5-iminodaunorubicin in human colon carcinoma cells.

The quinone-modified anthracycline, 5-iminodaunorubicin, which does not spontaneously generate free radicals, was compared to Adriamycin on the basis of DNA-protein crosslink-associated single-strand breakage, cell lethality, and the pharmacokinetics of drug uptake and efflux in human colon carcinoma cells in culture. At equivalent cytocidal concentrations, 5-iminodaunorubicin produced more single-strand breakage of DNA than Adriamycin after a 2-hr treatment interval, but the DNA scission produced by 5-iminodaunorubicin rapidly disappeared after drug removal. The kinetics of DNA breakage correlated with the rapid rates of uptake and efflux of 5-iminodaunorubicin in comparison to Adriamycin. These data emphasize the importance of the cellular pharmacokinetics of anthracyclines in relation to their cytocidal and DNA damaging properties. Moreover, the induction of equivalent single-strand breakage of DNA by similar intracellular concentrations of both drugs suggests that the free radical properties of Adriamycin are not involved in DNA scission.

Relationship of DNA strand breakage produced by bromodeoxyuridine to topoisomerase II activity in Bloom-syndrome fibroblasts.

Cells from patients with Bloom syndrome, a cancer-prone disorder with cutaneous photosensitivity and spontaneous chromosome breakage, exhibit an abnormally increased number of sister-chromatid exchanges following treatment with 5-bromodeoxyuridine (BrdU). This effect has been postulated to be mediated by abnormal topoisomerase II activity. We used alkaline elution to measure DNA single-strand breakage following prolonged exposure to BrdU. Five-day exposure to BrdU produced equal numbers of alkali-labile sites in normal and Bloom-syndrome fibroblasts. These breaks were not protein-associated but were produced by alkali. Treatment with topoisomerase II inhibitors induced similar frequencies of DNA single-strand breaks in normal and Bloom-syndrome fibroblasts. These findings imply that BrdU incorporation into cellular DNA induces alkali-labile DNA lesions that are independent of topoisomerase II activity in Bloom and normal cells.