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Biomedical subjects

D Kerrigan

Publications and source records attributed to D Kerrigan.

50 records · Page 3Linked to original sources
PubMed1985

Effect of difluoromethylornithine, an inhibitor of polyamine biosynthesis, on the topoisomerase II-mediated DNA scission produced by 4'-(9-acridinylamino)methanesulfon-m-anisidide in L1210 murine leukemia cells.

Treatment of mouse leukemia L1210 cells with the polyamine biosynthesis inhibitor alpha-difluoromethylornithine (DFMO) increased the magnitude of the DNA scission produced by the DNA intercalator 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA). This enhanced DNA scission was protein concealed and protein associated, as was the m-AMSA-induced scission in cells unexposed to DFMO. The effect of DFMO required more than 6 hr to develop and was greater at 48 hr than at 24 hr of exposure to DFMO. Exogenously added putrescine partially reversed the effects of DFMO, while exerting no effect on m-AMSA-induced DNA scission in cells unexposed to DFMO. The cellular uptake of [14C]-m-AMSA was the same in DFMO-treated or untreated cells. The DNA scission and DNA-protein cross-linking produced by m-AMSA appear to represent the stabilization of an intermediate in the normal cycle of topoisomerase II function (Nelson, E.M., Tewey, K.M., and Liu, L.F., Proc. Natl. Acad. Sci. USA, 81: 1361-1365, 1984). Since polyamine depletion appears to affect the magnitude of this effect in cells, and since polyamines can alter topoisomerase II function in vitro, polyamines may be involved in topoisomerase function in vivo either directly or through secondary effects, such as alterations of the conformation of chromatin, the intracellular site at which topoisomerase acts.

Aminoacridines↗
PubMed1984

Enhancement of the DNA breakage and cytotoxic effects of intercalating agents by treatment with sublethal doses of 1-beta-D-arabinofuranosylcytosine or hydroxyurea in L1210 cells.

4'-(9-Acridinylamino)methanesulfon-m-anisidide (m-AMSA) and other DNA intercalating agents produce protein-associated DNA strand breaks, the formation of which are mediated by topoisomerase-like chromosomal proteins. As topoisomerases would be expected to be most active during DNA replication, DNA synthesis inhibitors may alter the sensitivity of cellular DNA to intercalator-induced scission. We report that treatment of L1210 cells with 1-beta-D-arabinofuranosylcytosine (ara-C) (0.1 microM) or hydroxyurea (HU) (0.1 mM) for 18 hr resulted in a 2- to 2.4-fold enhancement of m-AMSA-induced protein-associated DNA single-strand breaks and DNA-protein cross-links as measured by alkaline elution. This enhancement was dependent on the duration of ara-C or HU treatment as well as on the concentration of ara-C or HU. Enhancement did not correlate with any alteration in cellular uptake of intercalator or with ara-C- or HU-induced alterations in the DNA synthetic rate. The DNA within nuclei isolated from ara-C- or HU-treated cells also displayed an enhanced susceptibility to m-AMSA-induced scission. There was a correlation between enhanced single-strand break formation and recruitment of cells into S-phase as well as between single-strand break formation and the production of a hypomethylated state of cellular DNA. Concurrent with the enhancement of m-AMSA-induced cellular DNA effects was a synergistic effect on m-AMSA cytotoxicity by ara-C or HU. This enhancement of intercalator effects was also found for the intercalator Adriamycin. We propose that these sublethal concentrations of ara-C and HU alter chromatin structure possibly via DNA hypomethylation and/or altered DNA-histone interactions so that intercalator-induced DNA effects are enhanced. Alternatively, the topoisomerase-like activity involved in intercalator-induced, protein-associated DNA break production may be increased in the nuclei of ara-C- or HU-treated cells.

Aminoacridines↗
PubMed1983

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.

Cell Survival↗
PubMed1983

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.

Animals↗
PubMed1983

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 Fanconi's 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.

Ataxia Telangiectasia↗
PubMed1983

Protein-associated intercalator-induced DNA scission is enhanced by estrogen stimulation in human breast cancer cells.

Estrogen-responsive human breast cancer cells (MCF-7) displayed a higher frequency of intercalator-induced protein-associated DNA scission after treatment with 17 beta-estradiol (E2) than did cells that had not received estrogen treatment. This effect was dependent on estrogen concentration (maximum enhancement at approximately equal to 1 nM E2) and time (maximum effect seen approximately equal to 24 hr after E2 addition). Human breast cancer cells lacking estrogen receptors did not display the enhanced response. Antiestrogens produced a slight decrease in intercalator-induced DNA scission, whereas insulin produced an enhanced effect. The DNA breaks produced by the intercalators 5-iminodaunorubicin and 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) in these cells were undetectable without enzymatic deproteinization of cell lysates prior to quantification by alkaline elution. Intercalator-induced DNA-protein crosslinking also was enhanced in E2-treated MCF-7 cells. Studies with m-[14C]AMSA revealed no estrogen-associated increases in drug uptake. The data suggest that E2 treatment, either by specifically and directly increasing active transcription in chromatin or through secondary effects on DNA that accompany alterations in cell growth or cell cycle distribution, alters the susceptibility of DNA to intercalator-induced protein-associated DNA scission. If this enhanced protein-associated scission is selectively localized to transcriptionally active chromatin, the adsorption of the DNA-bound proteins to membrane filters (DNA-protein crosslinking) may allow identification and isolation of estrogen-regulated gene sequences.

Breast Neoplasms↗
PubMed1983

Effects of dimethyl sulfoxide and thiourea upon intercalator-induced DNA single-strand breaks in mouse leukemia (L1210) cells.

The free radical scavengers, dimethyl sulfoxide (Me2SO) and thiourea, were used to assess the role of free radicals in the production of intercalator-induced DNA breaks and cytotoxicity in mouse leukemia L1210 cells. Both agents decreased X-ray break production, and this decrease was comparable in magnitude to the degree of inhibition of X-ray-induced cell killing. By contrast, Me2SO increased the DNA breaks produced by the intercalators, Adriamycin, 5-iminodaunorubicin, and 4'-(9-acridinylamino)methanesulfon-m-anisidide. This was not due to an enhancement of Adriamycin or 4'-(9-acridinylamino)methanesulfon-m-anisidide uptake by Me2SO. Strand break production by intercalators was decreased by thiourea. This was not due to an inactivation of the intercalators or to a decrease of Adriamycin or 4'-(9-acridinylamino)methanesulfon-m-anisidide uptake by thiourea. Experiments using nucleoid sedimentation to assess the DNA linking number and domain size from cells treated with Me2SO and thiourea indicated that these chemicals alter chromatin structure in a fashion which may account for effects on intercalator-induced DNA scission. The alterations in intercalator-induced DNA scission were not accompanied by corresponding alterations in cytotoxicity, thus dissociating intercalator-induced strand break production from lethality and the mechanism of X-ray break production.

Animals↗
PubMed1982

Cooperative sequestration of m-AMSA in L1210 cells.

The anticancer drug 4'-(9-acridinylamino)-methanesulfon-m-anisidide (m-AMSA) is known to bind to DNA by intercalation and to produce protein-associated DNA strand breaks in cells. Previous work [Zwelling et al., Biochemistry 20, 6553 (1981)] had shown that m-AMSA is in rapid equilibrium between extracellular and intracellular compartments, and that the DNA strand breaks exist in a steady state of rapid formation and resealing. The current work reports an unusual uptake phenomenon of m-AMSA by mouse leukemia L1210 cells that occurs at higher drug concentrations than previously studied. The new uptake phenomenon was characterized by cooperativity, hysteresis, irreversibility, saturability, slowness and temperature dependence. It is concluded that m-AMSA concentrations above a critical value can initiate the irreversible sequestration of m-AMSA into a new phase, probably in an extranuclear compartment of the cell, from which the drug has no access to the nuclear DNA and probably does not contribute to cytotoxicity.

Allosteric Regulation↗
PubMed1982

Formation and resealing of intercalator-induced DNA strand breaks in permeabilized L1210 cells without the stimulated synthesis of poly(ADP-ribose).

DNA strand breaks produced by damaging agents such as x-ray generally stimulate poly(adenosine diphosphoribose) (ADP-R) synthesis in mammalian cells. DNA intercalating agents induce the formation of strand breaks which are unusual in that they are associated with tightly or covalently bound protein. In order to determine whether the intercalator-induced strand breaks are associated with poly-(ADP-R) synthesis, L1210 cells were treated with the intercalating agent, 4'-(9-acridinylamino)methanesulfon-m-anisidide. Poly(ADP-R) synthesis, measured by [3H]NAD incorporation following cell permeabilization, was enhanced in x-irradiated cells, but not in cells exposed to 4'-(9-acridinylamino)methanesulfon-m-anisidide at doses which produced equivalent strand breaks frequencies. The permeabilized cell system did not support DNA synthesis and x-ray-induced strand breaks did not reseal. The intercalator-induced strand breaks, however, resealed within 10 min. Hence, the strand breaks observed in intercalator-treated cells may not constitute DNA damage in the usual sense. The resealing of intercalator-induced DNA breaks in the absence of DNA or poly(ADP-R) synthesis is unique among chemical or physical agents which produce DNA scissions.

Aminacrine↗
PubMed1982

Antibodies elicited against cis-diamminedichloroplatinum(II)-modified DNA are specific for cis-diamminedichloroplatinum(II)-DNA adducts formed in vivo and in vitro.

Rabbit antiserum elicited against calf thymus DNA modified to 4.4% (Pt drug/nucleotide ratio = 0.044) with the antitumor drug cis-diamminedichloroplatinum(II) (cis-DDP) contains antibodies specific for the Pt-modified DNA immunogen as well as for Pt-DNA adducts formed in both cultured mouse leukemia L1210 cells and in L1210 cells from the ascites fluid of tumor-bearing mice exposed to cis-DDP. Pt-modified DNA was electrostatically complexed to methylated bovine serum albumin and injected into rabbits. Early bleedings of the derived antiserum were used to establish a competitive enzyme-linked immunosorbent assay (ELISA), which demonstrated specificity for the Pt-modified DNA but not for DNA or the Pt drug alone. In the ELISA, 50% inhibition occurred at a concentration of 0.5 nM Pt (on DNA) as determined by atomic absorption spectroscopy. This value corresponds to a lower limit of detectability of one adduct in 10(7) nucleotides, with 50 micrograms of sample DNA added per microtiter well. DNA isolated from cultured mouse L1210 cells exposed to increasing doses of the Pt drug was found by ELISA to contain from 0.2 to 10.0 fmol of Pt adduct per microgram of DNA. These levels remained stable for up to 4 hr after a 1-hr drug treatment, during which time DNA interstrand crosslinks developed. Thus, the antiserum appears not to be specific for DNA interstrand crosslinks. DNAs from L1210 cells exposed to trans-diamminedichloroplatinum(II) and L-phenylalanine mustard were not recognized in the ELISA. DNA prepared from the ascites cells of mice bearing the L1210 tumor 5 hr after injection of cis-DDP was found to contain about 2 fmol of Pt per microgram of DNA. This work establishes that cis-DDP-DNA adducts prepared in vitro are relevant to the in vivo binding of the Pt drug to its biological target, DNA, and opens new avenues for studying the mechanism of action of the Pt anticancer drugs.

Animals↗
PubMed1982

Cytotoxicity and DNA strand breaks by 5-iminodaunorubicin in mouse leukemia L1210 cells: comparison with adriamycin and 4'-(9-acridinylamino)methanesulfon-m-anisidide.

The effects upon cellular DNA and cytotoxicity produced by the anthracyclines 5-iminodaunorubicin and Adriamycin were studied in mouse leukemia L1210 cells. 5-Iminodaunorubicin produced protein-concealed DNA strand breaks as measured by alkaline elution as had other intercalators including Adriamycin. 5-Iminodaunorubicin produced DNA breaks more efficiently than did Adriamycin despite a lower potency for free radical formation. Many of the 5-iminodaunorubicin breaks measured in this assay may arise from apposed single-strand breaks (i.e., double-strand breaks). 5-Iminodaunorubicin produced breaks which disappeared within 1 to 2 hr following drug removal and were in this way similar to the breaks produced by the acridine intercalator 4'-(9-acridinylamino)methanesulfon-m-anisidide. Adriamycin produced more persistent breaks. Despite similarities in the kinetics of break disappearance, 5-iminodaunorubicin produced greater cytotoxicity than did 4'-(9-acridinylamino)methanesulfon-m-anisidide when compared at doses producing equal single-strand or double-strand breaks. Differences in the ratio of single-strand breaks to double-strand breaks and the associated cytotoxicity for 5-iminodaunorubicin and 4'-(9-acridinylamino)methanesulfon-m-anisidide indicate that a different mechanism is probably involved in the DNA break production by each agent. Differences between the cytotoxicity associated with the DNA break production by two agents with similar break disappearance kinetics indicate that intercalator-induced DNA breaks cannot be a uniformly lethal DNA lesion.

Aminoacridines↗