OBJECTIVETo explore the role of phospholipase C-gamma1 (PLC-gamma1) signaling pathway in H(2)O(2)-induced apoptosis of PC12 cells.

METHODSPC12 cells were exposed to 50 micromol/L H(2)O(2) after pretreatment with 10 micromol/L U73122, a specific PLC-gamma1 inhibitor. Hoechst/PI double staining was performed to observe the morphological changes of the cells under light microscope. MTT assay was used to evaluate the cell viability, and the percentage of apoptotic cells was analyzed by flow cytometry. DNA fragmentation assay was carried out to characterize the cell apoptosis.

RESULTSAfter inhibition of the PLC-gamma1 signaling pathway with 10 micromol/L U73122, PC12 cells showed obvious apoptotic morphology, the viable cells decreased significantly, and the percentage of apoptotic cells rose to 35.7%. PC12 cells treated with U73122 presented with a distinct DNA ladder on electrophoresis resulting from DNA cleavage in the apoptotic cells.

CONCLUSIONPLC-gamma1 signaling pathway plays an important protective role in H(2)O(2)-induced PC12 cell apoptosis.

Animals ; Apoptosis ; drug effects ; Estrenes ; pharmacology ; Hydrogen Peroxide ; pharmacology ; PC12 Cells ; Phospholipase C gamma ; antagonists & inhibitors ; metabolism ; Pyrrolidinones ; pharmacology ; Rats ; Signal Transduction

BACKGROUNDIn most colorectal carcinomas, the level of phospholipase C (PLC)-gamma 1 expression is greatly elevated. Increased expression of PLC-gamma 1 may play an important role in colon carcinogenesis, but the mechanism is not well known. The aim of this study was to evaluate the role of PLC-gamma 1 in colon carcinogenesis by using recombinant lentivirus that stably suppressed the PLC-gamma 1 expression in human colorectal carcinoma LoVo cells.

METHODSRecombinant lentivirus producing PLC-gamma 1 siRNA were prepared. After LoVo cells were transduced by each lentivirus, stably transduced cells were selected by Blasticidin. The protein and mRNA expression of PLC-gamma 1 were examined by Western-blot and reverse transcription-polymerase chain reaction (RT-PCR) analysis, and the effects of the lentivirus on the cell adhesion, migration and apoptosis were analyzed.

RESULTSStable LoVo cell line deficient in PLC-gamma 1, was established. Notably, PLC-gamma 1 was silenced without affecting the levels of other subtypes of PLC so that the role of PLC-gamma 1 in colon carcinogenesis could be examined. Silencing of endogenous PLC-gamma 1 resulted in efficient inhibition of the adhesion and migration of LoVo cells in vitro and a great increase of 5-fluorouracil induced apoptosis (30%-40%) of LoVo cells.

CONCLUSIONSPLC-gamma 1 may play an important role in metastasis and anti-apoptosis in human colorectal carcinomas.

Apoptosis ; drug effects ; Cell Adhesion ; Cell Line, Tumor ; Colorectal Neoplasms ; pathology ; therapy ; Fluorouracil ; pharmacology ; Humans ; Laminin ; antagonists & inhibitors ; genetics ; Lentivirus ; genetics ; Phospholipase C gamma ; antagonists & inhibitors ; genetics ; physiology ; RNA, Small Interfering ; therapeutic use

OBJECTIVETo explore the role of phospholipase C-gamma1 (PLC-gamma1) in tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis of human glioma SWO cells.

METHODSThe PLC-gamma1 pathway was blocked by U73122 in SWO cells, and the inhibitory effect of TNF-alpha on SWO glioma cell proliferation with or without U73122 treatment was investigated by MTT assay. The cell apoptosis induced by TNF-alpha along or in combination with U73122 was detected by flow cytometry with PI staining. The expression of caspase-3 and Bcl-2 was detected by Western blotting.

RESULTS AND CONCLUSIONU73122 can sensitize SWO glioma cells to TNF-alpha-induced apoptosis. Blocking the PLC-gamma1 pathway may not induce apoptosis of SWO glioma cells, but can sensitize SWO glioma cells to small-dose TNF-alpha-induced apoptosis, the mechanism of which may involve down-regulation of bcl-2.

Apoptosis ; drug effects ; Blotting, Western ; Caspase 3 ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Dose-Response Relationship, Drug ; Down-Regulation ; drug effects ; Estrenes ; pharmacology ; Flow Cytometry ; Glioma ; enzymology ; pathology ; Humans ; Phosphodiesterase Inhibitors ; pharmacology ; Phospholipase C gamma ; antagonists & inhibitors ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Pyrrolidinones ; pharmacology ; Signal Transduction ; drug effects ; Tumor Necrosis Factor-alpha ; pharmacology

Phosphoinositide-specific phospholipase C-gamma1 (PLC-gamma1) has two pleckstrin homology (PH) domains: an amino-terminal domain (PH1) and a split PH domain (PH2). Here, we show that overlay assay of bovine brain tubulin pool with glutathione-S-transferase (GST)-PLC-gamma1 PH domain fusion proteins, followed by matrix-assisted laser-desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), identified 68-kDa neurofilament light chain (NF-L) as a binding protein of amino-terminal PH domain of PLC-gamma1. NF-L is known as a component of neuronal intermediate filaments, which are responsible for supporting the structure of myelinated axons in neuron. PLC-gamma1 and NF-L colocalized in the neurite in PC12 cells upon nerve growth factor stimulation. In vitro binding assay and immunoprecipitation analysis also showed a specific interaction of both proteins in differentiated PC12 cells. The phosphatidylinositol 4, 5-bisphosphate [PI(4,5)P2] hydrolyzing activity of PLC-gamma1 was slightly decreased in the presence of purified NF-L in vitro, suggesting that NF-L inhibits PLC-gamma1. Our results suggest that PLC-gamma1-associated NF-L sequesters the phospholipid from the PH domain of PLC-gamma1.
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Rats ; Protein Interaction Mapping ; Protein Biosynthesis/drug effects ; Protein Binding/drug effects ; Phosphoproteins/chemistry/*metabolism ; Phospholipase C gamma/antagonists & inhibitors/chemistry/*metabolism ; Phosphatidylinositol 4,5-Diphosphate/metabolism ; Peptides/chemistry/metabolism ; PC12 Cells ; Neurofilament Proteins/chemistry/*metabolism ; Nerve Growth Factor/pharmacology ; Molecular Weight ; Molecular Sequence Data ; Microtubules/metabolism ; Microscopy, Fluorescence ; Isoenzymes/metabolism/pharmacology/physiology ; Glutathione Transferase/metabolism ; Blotting, Far-Western ; Blood Proteins/chemistry/*metabolism ; Binding Sites ; Animals ; Amino Acid Sequence