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Our previous study showed that transmembrane TNF-α (TM-TNF-α) had broader tumoricidal spectrum than secretory TNF-α (s-TNF-α). This study examined the difference between the two kinds of TNF-α in inducing cells and the relationship between the apoptosis induced by TM-TNF-α and the cell cycle. Bioassay was employed to compare the cytotoxic effect of two kinds of TNF-α on cell lines L-929 and HepG2. TUNEL was used to detect apoptosis and the TdT and PI co-stainin gwere used for determining the phase of apoptotic cells. Our results showed that TM-TNF-α could kill not only s-TNF-sensitive L929 cells but also s-TNF-tolerant HepG2 cells. TM-TNF-α predominantly induced apoptosis while s-TNF could induce both apoptosis and necrosis. The apoptosis of L-929 cells induced by TM-TNF-α mainly occurred in S phase and the apoptosis of HepG2 predominantly took place in G1 phase. It is concluded that the cytotoxic effects of the two TNF differ substantially.Since TM-TNF-α works locally, mainly induces apoptosis and has broader anti-tumor spectrum, it may be more effective for the treatment of tumor than s-TNF.
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Objective To study the apoptosis and its molecular mechanism of MCF-7 cells induced by ?-3 polyunsaturated fatty acid(?-3 PUFA)combined with the product of exogenous transmembrane TNF-?(tmTNF-?) gene.Methods The tmTNF-? eukaryotic expression vector containing the PPRE-tk promoter was transfected into the human breast cancer MCF-7 cells.The effects of ?-3 PUFA and/or exogenous tmTNF-? on the cell proliferation and apoptosis were measured by MTT and DNA ladder assay.The activity of caspase-8 was examined by using special fluorescence substrate,and the expression of caspases(1,8 and 9) were analyzed by RT-PCR and Western blotting.The inhibitor of caspases was used to confirm the function of caspase.Results In 6.0?10~(-5)mol/L ?-3 PUFA-treated MCF-7 cells,only the growth suppression was found.In transfected MCF-7 cells after treated with ?-3 PUFA,not only the proliferation capacity was decreased but the DNA ladder was detectable.The expression changes of caspases(1,8 and 9) and caspase-8 activity were obvious in MCF-7 transfected cells treated with ?-3 PUFA.Growth inhibition and apoptosis induced by ?-3 PUFA and tmTNF-? were partly prevented by the special caspase inhibitor.Conclusion These results suggested that up-regulated expression and activity of caspase might promote MCF7 cells apoptosis induced by ?-3 PUFA and exogenous tmTNF-?,indicating that ?-3 PUFA and exogenous tmTNF-? could cooperate in inhibition of the MCF-7 cell growth and induction of apoptosis.caspase network pathway may play a key role in these processes.
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Objective:To study the effects of PTK on the respiratory burst and release of NO by neutrophils in response to transmembrane TNF-?( TM-TNF-?) and secreted TNF-? ( S-TNF-?). Methods: The effects of PTK inhibitor on the functions of neutrophils caused by both forms of TNF-? was detected with the chemiluminescence and the nitrate reductase assay.The tyrosine phosphorylation of neutrophils induced by the two forms of TNF-? was compared by Western blotting analysis. Results: The PTK inhibitor genistein (50 ?mol/L) was found not only to depress the respiratory burst of neutrophils induced by S-TNF-?( P
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Transmembrane TNF-? is an integral protein expressed on the surface of avtivated monocytes/macrophages. In order to define the interrelation between TM-TNF and biomembrane, we used an in vitro translation system and microsomal membranes to engender expression models of two types of TM-TNF; TM-TNF anchored to microsomes and naked 26kD TNF without membranes. When the cytotoxic activity of these two types of TNF was compared, the synthesized 26kD TNF was found to exert its effects only in the presence of microsomal membranes. With the use of indirect immunofluorescence technique, we further studied the binding of the in vitro translated 26kD TNF with TNFR on the membrane of HL60 cells. It was found that only TM-TNF attached to the microsomes could bind effectively with TNFR, while naked 26kD TNF could not. These results suggest that binding with biomembrane may be the prerequisite for 26kD TNF to fold properly for displaying its biological effects. In case 26kD TNF is freed from membrane, it can no more bind with TNFR, there by leading to loss of its cytotoxic effect.
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Objective:To determine the region of human transmembrane tumor necrosis factor alpha (TM TNF?),essential for cytotoxic activity against mouse L929 cell.Methods:Single amino acid substituted TM TNF? mutant proteins(muteins) were produced by in vitro transcription linked translation techniques.An expression cDNA for TM TNF? was site directed mutagenized by recombinant PCR.7 single amino acid substituted TM TNF? muteins were generated and assayed for cytotoxic activity.Results:The cytotoxic activities of TM TNF? muteins,eg,TM TNF? -71/Lys was
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Objective To construct the transmembrane TNF-? eukaryotic expression vector in n-3 PUFA dependant manner. Methods PPRE-tk sequence was designed and artificially synthesized, and then inserted it into pcDNA3-TNF( ? 1-12)WB plasmid to construct eukaryotic expression vector pPPRE-tk-tmTNF-? by gene recombine techniques. The tmTNF-? protein expression level was observed in MCF-7 transfected cells incubated with EPA by immunofluorescence technique. Results pPPRE-tk-tmTNF-? expression vector was constructed successfully and identified by agarose gel electrophoretic analysis and nucleotide sequence analysis. EPA could increase tmTNF expression levels in time- and dose-dependant manners. Conclusion tmTNF-? expression vector regulated by n-3 PUFA is successfully constructed.
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Objective:To study the effect of ?-3 PUFA on the proliferation and apoptosis of tumor cells transfected with transmembrane TNF? (tmTNF?) expression vector containing peroxisome proliferator responsive element (PPRE). Methods:The tmTNF? expression vector was transfected into HL-60 and MCF-7 tumor cells. The effects of ?-3PUFA and/or exogenous tmTNF? on cell proliferation, cell cycle distribution and apoptosis were measured by growth curve,flow cytometry and DNA ladder assay. Results: The production of exogenous tmTNF?gene was located in the cell membrane. After treatment with?-3 PUFA, the expression of exogenous tmTNF? in transfectant was significantly increased. The proliferation was decreased in HL-60 and MCF-7 cells treated with 6.0?10-5 mol/L ?-3 PUFA,but cell cycle was arrested at G0/G1 and the DNA ladder was detectable only in HL-60 cells. These changes were more obvious in transfected cells than non-transfected cells. Conclusion:?-3 PUFA and the product of exogenous tmTNF? gene induced by ?-3 PUFA could inhibit tumor cell growth more effectually,and induction of apoptosis may play a key role in this process.