Oncogenic ras mediates apoptosis in response to protein kinase C inhibition through the generation of reactive oxygen species.
Ras is a well established modulator of apoptosis. Suppression of protein kinase C (PKC) activity can selectively induce apoptosis in cells expressing a constitutively activated Ras protein. We wished to determine whether reactive oxygen species serve as an effector of Ras-mediated apoptosis. Ras-transformed NIH/3T3 cells contained higher basal levels of intracellular H(2)O(2) compared with normal NIH/3T3 cells, and PKC inhibition up-regulated ROS to 5-fold greater levels in Ras-transformed cells than in normal cells. Treatment with N-acetyl-l-cysteine reduced both the basal and inducible levels of intracellular H(2)O(2) in NIH/3T3-Ras cells and antagonized the induction of apoptosis by PKC inhibition. Culturing NIH/3T3-Ras cells in low oxygen conditions, which prevents ROS generation, also inhibited the apoptotic response to PKC inhibition. These results suggest that reactive oxygen species are necessary as downstream effectors of the Ras-mediated apoptotic response to PKC inhibition. However, the generation of ROS alone is not sufficient to induce apoptosis in Ras-transformed cells because inhibition of cell cycle progression prevented the induction of apoptosis in NIH/3T3-Ras cells without inhibiting the generation of intracellular H(2)O(2) observed after PKC inhibition. These findings suggest that continued cell cycle progression of Ras-transformed cells during PKC inhibition is also necessary for the induction of apoptosis.
Liou
J S
JS
Boston University School of Medicine, Cancer Research Center, Boston, Massachusetts 02118, USA.
Chen
C Y
CY
Chen
J S
JS
Faller
D V
DV
D016475:3T3 Cells; D000111:Acetylcysteine / Q000494:pharmacology; D000818:Animals; D000935:Antifungal Agents / Q000494:pharmacology; D017209:Apoptosis; D002087:Butyrates / Q000494:pharmacology; D002453:Cell Cycle / Q000187:drug effects; D002469:Cell Separation; D002470:Cell Survival / Q000187:drug effects; D003524:Cyclosporins / Q000494:pharmacology; D053938:DNA Fragmentation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D005452:Fluoresceins / Q000494:pharmacology; D016166:Free Radical Scavengers / Q000494:pharmacology; D005995:Glyceryl Ethers / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000378:metabolism; D006877:Hydroxamic Acids / Q000494:pharmacology; D051379:Mice; D008938:Mitosis / Q000187:drug effects; D010100:Oxygen / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors* / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection; D015854:Up-Regulation; D018631:ras Proteins / Q000502:physiology*
C110400:2',7'-dichlorodihydrofluorescein diacetate; D000935:Antifungal Agents; D002087:Butyrates; D003524:Cyclosporins; D004791:Enzyme Inhibitors; D005452:Fluoresceins; D016166:Free Radical Scavengers; D005995:Glyceryl Ethers; D006877:Hydroxamic Acids; D017382:Reactive Oxygen Species; C059039:1-O-hexadecyl-2-O-methylglycerol; C012589:trichostatin A; C100000:cyclosporin C; D006861:Hydrogen Peroxide; D011493:Protein Kinase C; D018631:ras Proteins; C019475:2-methylbutanoic acid; D010100:Oxygen; D000111:Acetylcysteine