Overexpression of the transgene for manganese superoxide dismutase (MnSOD) in 32D cl 3 cells prevents apoptosis induction by TNF-alpha, IL-3 withdrawal, and ionizing radiation.
OBJECTIVE
Stabilization of the mitochondria in IL-3-dependent hematopoietic progenitor cell line 32D cl 3 by overexpression of the transgene for manganese superoxide dismutase (MnSOD) prior to ionizing radiation prevents apoptosis. We now demonstrate that overexpression of the MnSOD transgene also protects 32D cl 3 cells from apoptosis caused by exposure to tumor necrosis factor-alpha (TNF-alpha) or withdrawal of interleukin (IL)-3.


METHODS
The hematopoietic progenitor cell line, 32D cl 3, and subclones overexpressing the human MnSOD transgene, 1F2 or 2C6, were radiated to 1000 cGy or were exposed to TNF-alpha (0 to 100 etag/mL) or were subjected to IL-3 withdrawal. The cells were then examined at several time points for DNA strand breaks using a comet assay, depolarization of the mitochondrial membrane, activation of caspase-3, PARP cleavage, and apoptosis, and also for changes in cell cycle distribution.


RESULTS
Overexpression of the transgene for MnSOD resulted in increased survival following exposure to radiation, exposure to TNF-alpha, or IL-3 withdrawal. The cell lines overexpressing MnSOD (1F2 or 2C6) displayed decreased radiation-induced, TNF-alpha-induced, or IL-3 withdrawal-induced mitochondrial membrane permeability, caspase-3 and PARP activation, and apoptosis.


CONCLUSIONS
Overexpression of the human MnSOD transgene in 32D cl 3 cells results in stabilization of the mitochondria and reduction in radiation-, TNF-alpha-, or IL-3 withdrawal-induced damage. Thus, MnSOD stabilization of the mitochondrial membrane is relevant to reduction of apoptosis by several classes of oxidative stress inducers.
Epperly
Michael W
MW
Department of Radiation Oncology, University of Pittsburgh Cancer Institute, Hillman Cancer Center, 200 Lothrop Street, Pittsburgh, PA 15213, USA.
Bernarding
Michael
M
Gretton
Joan
J
Jefferson
Mia
M
Nie
Suhua
S
Greenberger
Joel S
JS
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000528:radiation effects; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D004249:DNA Damage; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000201:enzymology; D006801:Humans; D007377:Interleukin-3 / Q000494:pharmacology; D007425:Intracellular Membranes / Q000187:drug effects / Q000528:radiation effects; D051379:Mice; D008928:Mitochondria / Q000201:enzymology / Q000648:ultrastructure; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D021381:Protein Transport; D011506:Proteins / Q000378:metabolism; D011839:Radiation, Ionizing; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D019076:Transgenes; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D007377:Interleukin-3; D011506:Proteins; D014409:Tumor Necrosis Factor-alpha; D013482:Superoxide Dismutase; C499321:Parp1 protein, mouse; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; C505407:CASP3 protein, human; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases