Cell death induced by anticancer agents involves many targets but in each case, a subsequent step involves endonuclease-mediated digestion of cellular DNA. This DNA digestion is characteristic of """"""""programmed cell death"""""""" or """"""""apoptosis"""""""" and is considered to represent an essential step in the process of cell death. The timing of apoptotic DNA degradation after equitoxic treatment varies with agent occurring within 30 min after hyperthermia but 3 days after cisplatin. In the latter case, death occurs at the G2/M phase of the cell cycle. The first goal of this proposal is to purify and characterize an acidic endonuclease, identified as deoxyribonuclease II, that has been implicated in cell death after hyperthermia. The purification has been achieved and a partial amino acid sequence obtained. Based on this sequence, oligonucleotides will be synthesized and used to isolate the cDNA. Antibodies will also be raised. The cDNA and antibodies will be used as probes to investigate the normal function of this endonuclease and its contribution to cell death: one approach will involve suppression or enhancement of endonuclease activity. The role of intracellular acidification after lethal insults will be investigated using a pH sensitive fluorescent dye. The structure of the cut ends of DNA will be investigated as a potential diagnosis of the activity of this endonuclease. Another goal is to establish a cause or effect relationship between cell death and the p34cdc2 kinase that regulates passage into mitosis. Assays will be performed to confirm that the kinase activity is suppressed during G2 arrest and to determine whether activity increases again as cells progress toward death. A cell line temperature sensitive for p34cdc2 kinase will be used to resolve the cause or effect relationship. Other experiments will investigate the role of the endonuclease and p34cdc2 in cell death following other insults including aphidicolin, and conditions considered as physiologic cell death (e.g. glucorticoid-mediated death of thymocytes). Subsequent experiments will investigate the regulation of the endonuclease and kinase. The outcome of these experiments may be the development of new strategies for drug development, for synergistic therapy, and for overcoming drug resistance.

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National Cancer Institute (NCI)
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Experimental Therapeutics Subcommittee 1 (ET)
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Dartmouth College
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Bates, Darcy J P; Danilov, Alexey V; Lowrey, Christopher H et al. (2013) Vinblastine rapidly induces NOXA and acutely sensitizes primary chronic lymphocytic leukemia cells to ABT-737. Mol Cancer Ther 12:1504-14
Albershardt, Tina C; Salerni, Bethany L; Soderquist, Ryan S et al. (2011) Multiple BH3 mimetics antagonize antiapoptotic MCL1 protein by inducing the endoplasmic reticulum stress response and up-regulating BH3-only protein NOXA. J Biol Chem 286:24882-95
Bates, Darcy J P; Salerni, Bethany L; Lowrey, Christopher H et al. (2011) Vinblastine sensitizes leukemia cells to cyclin-dependent kinase inhibitors, inducing acute cell cycle phase-independent apoptosis. Cancer Biol Ther 12:314-25
Salerni, Bethany L; Bates, Darcy J; Albershardt, Tina C et al. (2010) Vinblastine induces acute, cell cycle phase-independent apoptosis in some leukemias and lymphomas and can induce acute apoptosis in others when Mcl-1 is suppressed. Mol Cancer Ther 9:791-802