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ORIGINAL RESEARCH ARTICLE

Caspase-Dependent and -Independent Panc-1 Cell Death Due to Actinomycin D and MK 886 Are Additive but Increase Clonogenic Survival

Kenning M. Anderson^*,,1, Waddah Alrefai^*, Philip Bonomi^*, Thomas M. Seed, Pradeep Dudeja^¶, Yunming Hu^* and Jules E. Harris^*

^* Section of Medical Oncology, Departments of Medicine and
Biochemistry, Rush Medical College, Chicago, Illinois 60612;
Armed Forces Radiobiology Research Institute, Bethesda, Maryland 20889; and
^¶ Department of Physiology and West Side Veterans Administration Medical Center, University of Illinois, Chicago, Illinois 60612

In human panc-1 pancreatic cancer cells, actinomycin D (act D) induces a type 1 (apoptotic, extrinsic, death domain, receptor-dependent, and caspase-positive) form of programmed cell death (PCD) and MK 886, a 5-lipoxygenase inhibitor serving among other functions as a surrogate for increasing oxidative stress, a type 2 form, defined as an intrinsic, mitochondria-dependent, autophagic form of cellular suicide. Using both agents simultaneously should allow for examination of their interaction in cells able to express either form of PCD. Activation of both forms might result in synergistic, additive, null, or inhibitory effects on the reduction in proliferation, PCD, and clonogenicity of surviving cells. Co-culture of panc-1 cells with act D and MK 886, which both inhibit their proliferation, had an additive effect on increasing the development of these forms of PCD, as determined by morphology, a nucleosome assay, and flow cytometry. Initially, laddering on agarose detected with propidium iodide, present in act D, and act D plus MK 886-treated cells was partially obscured by randomly degraded DNA. With the use of the more sensitive SYBR green dye and reduced exposure of detached cells to 37°C, a limited laddering of DNA from MK 886-treated cells was also detected. Caspase activity was present in act-D-cultured cells but was absent in cells cultured with MK 886. Combined culture reduced caspase activity in act D-treated cells, consistent with interference from type 2 of type 1 PCD. Removal after 48 hr of act D or MK 886 allowed regrowth of residual cells, the latter agent to a greater extent than the former. In combination, the number of clones was increased compared with act D alone. These features distinguish two forms of PCD. In therapeutic settings in which the modes of cell death have not been identified, unintentional activation of several cellular suicide pathways with "crosstalk" between them occurs. Their intentional simultaneous activation and responses, as modulated by the history of cells in or out of cycle, could reduce the intended therapeutic outcome with survival of additional clonogenic cells due to various forms of mutual interference.

Key Words: pancreatic cancer • Panc-1 • Actinomycin D • MK 886 • programmed cell death type 1 and type 2