Robert M. Snapka

Inhibition of topoisomerase II by liriodenine

The cytotoxic oxoaporphine alkaloid liriodenine, isolated from Cananga odorata, was found to be a potent inhibitor of topoisomerase II (EC 5.99.1.3) both in vivo and in vitro. Liriodenine treatment of SV40 (simian virus 40)-infected CV-1 cells caused highly catenated SV40 daughter chromosomes, a signature of topoisomerase II inhibition. Strong catalytic inhibition of topoisomerase II by liriodenine was confirmed by in vitro assays with purified human topoisomerase II and kinetoplast DNA. Liriodenine also caused low-level protein-DNA cross-links to pulse-labeled SV40 chromosomes in vivo, suggesting that it may be a weak topoisomerase II poison. This was supported by the finding that liriodenine caused topoisomerase II-DNA cross-links in an in vitro assay for topoisomerase II poisons. Verapamil did not increase either liriodenine-induced protein-DNA cross-links or catalytic inhibition of topoisomerase II in SV40-infected cells. This indicates that liriodenine is not a substrate for the verapamil-sensitive drug efflux pump in CV-1 cells.

Exposure to camptothecin breaks leading and lagging strand simian virus 40 DNA replication forks

To better understand aberrant simian virus 40 DNA replication intermediates produced by exposure of infected cells to the anticancer drug camptothecin, we compared them to forms produced by S1 nuclease digestion of normal viral replication intermediates. All of the major forms were identical in both cases. Thus the aberrant viral replicating forms in camptothecin-treated cells result from DNA strand breaks at replication forks. Linear simian virus 40 forms which are produced by camptothecin exposure during viral replication were identified as detached DNA replication bubbles. This indicates that double strand DNA breaks caused by camptothecin-topoisomerase I complexes occur at both leading and lagging strand replication forks in vivo.

Two-dimensional agarose gel analysis of simian virus 40 DNA replication intermediates

Normal and aberrant simian virus 40 DNA (SV40) replication intermediates can be studied by high-resolution two-dimensional gel electrophoresis. Because the SV40 chromosome is often considered a model for the mammalian replicon, this experimental system can be used to understand how cytotoxic drugs and physical stressors affect DNA replication in mammalian cells. In this article we describe the use of two-dimensional neutral-alkaline and neutral-chloroquine gels in detail. In both systems, first-dimension neutral agarose gels are used to separate viral replication intermediates on the basis of their compactness. The second-dimension gel is then run at a 90° angle to the first. Alkaline second-dimension electrophoresis is carried out in a strongly denaturing, high-pH buffer. Denaturation of nicked or gapped DNA replication intermediates separates the pulse-labeled daughter DNA strands from the unlabeled parental strands. Alkaline second-dimension gels can reveal covalent or topological linkages between parental and daughter DNA strands. In second-dimension chloroquine gel electrophoresis, the electrode buffer contains the intercalating drug chloroquine. Chloroquine titrates out negative supercoils in superhelical DNA replication intermediates and introduces positive supercoils into covalently closed, relaxed intermediates. Because the basis of the separation in each dimension is known, the nature of natural or aberrant DNA replication intermediates can often be deduced from their two-dimensional gel behavior.

PCNA damage caused by antineoplastic drugs

Structurally diverse chemotherapeutic and chemopreventive drugs, including camptothecin, doxorubicin, sanguinarine, and others, were found to cause covalent crosslinking of proliferating cell nuclear antigen (PCNA) trimers in mammalian cells exposed to fluorescent light. This PCNA damage was caused by both nuclear and cytoplasmically localizing drugs. For some drugs, the PCNA crosslinking was evident even with very brief exposures to laboratory room lighting. In the absence of drugs, there was no detectable covalent crosslinking of PCNA trimers. Other proteins were photo-crosslinked to PCNA at much lower levels, including crosslinking of additional PCNA to the PCNA trimer. The proteins photo-crosslinked to PCNA did not vary with cell type or drug. PCNA was not crosslinked to itself or to other proteins by superoxide, hydrogen peroxide or hydroxyl radicals, but hydrogen peroxide caused monoubiquitination of PCNA. Quenching of PCNA photo-crosslinking by histidine, and enhancement by deuterium oxide, suggest a role for singlet oxygen in the crosslinking. SV40 large T antigen hexamers were also efficiently covalently photo-crosslinked by drugs and light. Photodynamic crosslinking of nuclear proteins by cytoplasmically localizing drugs, together with other evidence, argues that these drugs may reach the nucleoplasm in amounts sufficient to photodamage important chromosomal enzymes. The covalent crosslinking of PCNA trimers provides an extremely sensitive biomarker for photodynamic damage. The damage to PCNA and large T antigen raises the possibility that DNA damage signaling and repair mechanisms may be compromised when cells treated with antineoplastic drugs are exposed to visible light.

Nicotine Mediates Hypochlorous Acid-Induced Nuclear Protein Damage in Mammalian Cells

Activated neutrophils secrete hypochlorous acid (HOCl) into the extracellular space of inflamed tissues. Because of short diffusion distance in biological fluids, HOCl-damaging effect is restricted to the extracellular compartment. The current study aimed at investigating the ability of nicotine, a component of tobacco and electronic cigarettes, to mediate HOCl-induced intracellular damage. We report, for the first time, that HOCl reacts with nicotine to produce nicotine chloramine (Nic-Cl). Nic-Cl caused dose-dependent damage to proliferating cell nuclear antigen (PCNA), a nuclear protein, in cultured mammalian lung and kidney cells. Vitamin C, vitamin E analogue (Trolox), glutathione, and N-acetyl-l-cysteine inhibited the Nic-Cl-induced PCNA damage, implicating oxidation in PCNA damage. These findings point out the ability of nicotine to mediate HOCl-induced intracellular damage and suggest antioxidants as protective measures. The results also raise the possibility that Nic-Cl can be created in the inflamed tissues of tobacco and electronic cigarette smokers and may contribute to smoking-related diseases.

Drug-Induced Loss of Unstably Amplified Genes

Methotrexate-resistant R500 cells slowly lose amplified dihydrofolate reductase (dhrf) genes with biphasic kinetics when grown in the absence of methotrexate. Both phases of gene loss were markedly accelerated by subcytotoxic drug treatments. R500 cells were passed in low concentrations of cytotoxic drugs (inhibitors of ribonucleotide reductase, type I and type II topoisomerases, and polyamine synthesis). At each passage, relative dhfr gene copy number was determined by slot blot analysis. All of these drugs were able to induce rapid loss of dhfr gene dosage in the R500 cell population. The ability of these treatments to cause the rapid emergence of a cell population with substantially reduced dhfr gene dosage indicates that either the amplified genes or those cells with the highest levels of gene amplification are selectively targeted by low-level cytotoxic stress. The complex kinetics of amplified gene loss are suggestive of differential targeting of resistant cell subpopulations.

Ubiquitin-Family Modifications of Topoisomerase I in Camptothecin-Treated Human Breast Cancer Cells

Camptothecins kill mammalian cells by stabilizing topoisomerase I-DNA strand passing intermediates that are converted to lethal double strand DNA breaks in DNA replication fork collisions. Camptothecin-stabilized topoisomerase I-DNA cleavage intermediates in mammalian cells are uniquely modified by ubiquitin-family proteins. The structure, composition, and function of these ubiquitin-family modifications are poorly understood. We have used capillary liquid chromatography-nanospray tandem mass spectrometry to analyze the endogenous ubiquitin-family modifications of topoisomerase I purified from camptothecin-stabilized topoisomerase I-DNA cleavage complexes in human breast cancer cells. Peptides shared by SUMO-2 and SUMO-3 were abundant, and a peptide unique to SUMO-2 was identified. Ubiquitin was also identified in these complexes. No SUMO-1 peptide was detected in human topoisomerase I-DNA cleavage complexes. Identical experiments with purified SUMO paralogues showed that SUMO-1 was well digested by our protocol and that fragments were easily analyzed by LC-MS/MS. Spiking experiments with purified SUMO paralogues determined that we could detect as little as 0.5 SUMO-1 residue per topoisomerase I molecule. These results indicate that SUMO-1 is below this detection level and that SUMO-2 or a mixture of SUMO-2 and SUMO-3 predominates. SUMO-1 capping seems unlikely to be limiting the growth of SUMO-2/3 chains formed on camptothecin-stabilized topoisomerase I-DNA cleavage complexes.

Hypersensitivity to low level cytotoxic stress in mouse cells with high levels of DHFR gene amplification.

Low level cytotoxic stress greatly accelerates the loss of unstably amplified dihydrofolate reductase (dhfr) genes from methotrexate-resistant mouse cell lines. To understand this drug-induced loss of amplified genes, the highly methotrexate-resistant mouse R500 cell line was flow sorted into two subpopulations with higher and lower average dhfr gene dosage respectively. The subpopula-tion with higher levels of gene amplification was much more sensitive to low level cytotoxic stress as judged by both cloning efficiency and growth in the presence of low concentrations of cytotoxic drugs. These results suggest that high levels of gene amplification can confer hypersensitivity to cytotoxic Stressors such as anticancer drugs.

Bromodeoxyuridine photodamage in studies of UVA damage and the cell cycle.

Dear Editor: I would like to comment on the article by Girard and co-workers entitled “Inhibition of S-phase progression triggered by UVA-induced ROS does not require a functional DNA damage checkpoint response in mammalian cells” [1]. Their study involves the effects of ultraviolet A (UVA) on the cell cycle in the presence or absence of the ATM, ATR and p38 signaling pathways. The effects of UVA exposure on the cell cycle are assumed to be due to UVA absorption by cellular molecules or media components. In contrast to earlier flow cytometry studies of ultraviolet A (UVA) cell cycle effects [2,3], Girard et al. incorporate bromodeoxyuridine (BrdU) into cellular DNA before UVA exposure. BrdU is a type I photosensitizer capable of causing a variety damage to cellular molecules [4,5]. This raises the possibility of molecular damage due to BrdU photolysis in the flow cytometry studies of Girard et al. It cannot be assumed that the cell cycle effects observed are due only to UVA absorption by cellular molecules or medium components. The light source used by Girard et al. (described in their reference 40) has a substantial component at 313 nm, a wavelength at which UVA photo-nicking of BrdU substituted DNA is very efficient even in the absence of dyes [6]. The peak of the source used by Girard et al. is at 365 nm, where UVA photo-nicking of BrdU substituted DNA is about three orders of magnitude less [6], but the irradiance of the source at 365 nm is also about five orders of magnitude greater than at 313 nm. UVA irradiation of cells that have incorporated BrdU, using similar light sources, has been shown to be very mutagenic [7], and to inactivate DNA virus [8]. In addition to base damage and DNA strand breaks, there is a possibility that irradiation of BrdU substituted DNA at these wavelengths can cause DNA-DNA crosslinks [9] and DNA-protein crosslinks [10,11]. The possibility of several types of molecular damage from UVA photolysis of BrdU complicates the interpretation of the studies reported by Girard et al..

Abstract 2252: Mechanism of thymoquinone-induced cell death in glioblastoma multiforme.

Glioblastoma (GBM) is the most aggressive and common type of brain tumor in humans. Surgical resection followed by radiation and chemotherapy is the current standard treatment; however, due to the upregulation of several survival pathways, tumor recurrence is quite common with a median survival of 12 to 15 months. Therefore, there is an urgent need for more effective therapeutic strategies for the treatment of glioblastoma. Naturally occurring phytochemicals have steadily received much scientific attention, owing to the fact that many of these compounds have potent action against tumors. Thymoquinone (TQ) is the bioactive compound of the volatile oil extracted from the black seed, Nigella sativa. TQ has shown anti-oxidant, anti-inflammatory and anti-neoplastic actions with a selective cytotoxicity for human cancer cells compared to several normal cells. Here, we show that TQ can dose-dependently inhibit colony formation in three distinct glioblastoma cell lines, with the highly tumorigenic Gli36ΔEGFR displaying the greatest sensitivity to the anti-proliferative effects of TQ. Moreover, normal human astrocytes are far less sensitive to TQ-induced cell kill. The absence of both DNA fragmentation and PARP cleavage in U87 and Gli36ΔEGFR cells suggests that TQ does not activate apoptosis. We show that TQ does not change the levels of Beclin-1 expression, but does cause accumulation of LC3-II and p62 protein levels. This suggests that TQ blocks the late stages of autophagy. TQ-induced cell death can be partially rescued by co-treatment with a lysosomal cathepsin hydrolase inhibitor III. We also show that TQ induces persistent γ-H2AX activation and causes G2/M cell cycle arrest in GBM cells to a degree that correlates with the cells’ relative clonogenic sensitivity. Moreover, co-treatment with TQ and N-acetyl cysteine, a simple thiol nucleophile and antioxidant, prevented cell death, DNA double strand breaks, and cell cycle arrest. Citation Format: Ira O. Racoma, Robert M. Snapka, Altaf A. Wani. Mechanism of thymoquinone-induced cell death in glioblastoma multiforme. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2252. doi:10.1158/1538-7445.AM2013-2252