ABSTRACT
The activation of gene transcription by light is transient since light-dependent mRNA accumulation ceases after long exposures to light. This phenomenon, photoadaptation, has been observed in plants and fungi, and allows the perception of changes in light intensities. In the fungus Neurosporacrassa photoadaptation involves the transient binding of the photoresponsive White Collar Complex (WCC) to the promoters of light-regulated genes. We show that RCO-1 and RCM-1, the Neurospora homologs of the components of the yeast Tup1-Ssn6 repressor complex, participate in photoadaptation. Mutation in either rco-1 or rcm-1 result in high and sustained accumulation of mRNAs for con-10 and other light-regulated genes after long exposures to light. The mutation of rco-1 increased the sensitivity to light for con-10 activation and delayed synthesis and/or degradation of con-10 and con-6 mRNAs without altering the amount or the light-dependent phosphorylation of the photoreceptor WC-1. RCO-1 and RCM-1 are located in the Neurospora nuclei were they regulate gene transcription. We show that RCO-1 and RCM-1 participate in the light-transduction pathway of Neurospora and has a role in photoadaptation by repressing gene transcription after long exposures to light.
Subject(s)
Fungal Proteins/physiology , Gene Expression Regulation, Fungal , Neurospora crassa/physiology , Repressor Proteins/physiology , Signal Transduction , Transcription, Genetic , Cell Nucleus/chemistry , DNA-Binding Proteins/metabolism , Fungal Proteins/genetics , Fungal Proteins/metabolism , Light , Neurospora crassa/genetics , Phosphorylation , Repressor Proteins/genetics , Transcription Factors/metabolismABSTRACT
In a preliminary study, we found that benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD- fmk), unlike Boc-aspartyl(OMe)-fluoromethylketone (BocD-fmk), at usual dosage could not prevent genistein-induced apoptosis of p815 mastocytoma cells. This study was undertaken to reveal the mechanism underlying the incapability of zVAD-fmk in preventing this type of apoptosis. We observed that 14-3-3 protein level was reduced in genistein-treated cells and that BocD-fmk but not zVAD-fmk prevented the reduction of 14-3-3 protein level and the release of Bad from 14-3-3. We also demonstrated that truncated Bad to Bcl-xL interaction in genistein- treated cells was prevented by BocD-fmk but not by zVAD-fmk treatment. Our data indicate that BocD- fmk, compared to zVAD-fmk, has a certain preference for inhibiting 14-3-3/Bad signalling pathway. We also elucidated that this differential efficacy of BocD-fmk and zVAD-fmk resulted from the different effect in inhibiting caspase-6 and that co-treatment of zVAD-fmk and caspase-6 specific inhibitor substantially prevented genistein-induced apoptosis. Our data shows that caspase-6 plays a role on Bad/14-3-3 pathway in genistein-induced apoptosis of p815 cells, and that the usual dose of zVAD-fmk, in contrast to BocD-fmk, did not prevent caspase-6 acting on 14-3-3/Bad-mediated event.
Subject(s)
14-3-3 Proteins/metabolism , Apoptosis/drug effects , Benzyl Compounds/pharmacology , Caspase 6/metabolism , Enzyme Inhibitors/pharmacology , Hydrocarbons, Fluorinated/pharmacology , Signal Transduction , bcl-Associated Death Protein/metabolism , Amino Acid Chloromethyl Ketones/pharmacology , Animals , Caspase Inhibitors , Cell Line, Tumor , Genistein/pharmacology , Mastocytoma , Mice , Mitochondria/drug effects , Signal Transduction/drug effectsABSTRACT
Sulindac is one of the most widely studied nonsteroidal anti-inflammatory drugs in the prevention of colon cancer. Thus, from the viewpoint of colon cancer chemotherapy it is important to reveal the mechanism of sulindac-induced cell death. This study was undertaken to dissect the molecular mechanism underlying sulindac-induced apoptosis in human colon cancer cell line HT-29 (mutant p53), focusing on nuclear translocation of AIF, DFF and endonuclease G. On induction of apoptosis by sulindac, it was associated with decreased mitochondrial membrane potential, nuclear expression of active caspase-3, cleavage of poly(ADP-ribose) polymerase, translocation of mitochondrial proteins to the nucleus, and morphological evidence of nuclear condensation. However, sulindac led to only disintegration of nuclear DNA into high molecular weight DNA fragments of about 100-300 kbp as determined by a pulse-field gel electrophoresis, suggesting a predominantly AIF-mediated cell death process. In summary, our findings indicate that sulindac induces large-scale DNA fragmentation without oligonucleosomal DNA fragmentation. This result suggests that nuclear translocation of DFF and endonuclease G are not sufficient for the induction of oligonucleosomal DNA fragmentation in HT-29 cells.
