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Increase of MnSOD expression and decrease of JNK activity determine the TNF sensitivity in bcl2-transfected L929
1Department of Biochemistry, College of Science and Bioproducts Research Center, Yonsei University, Seoul, 120-749, Korea.
Abstract:
To investigate the protection mechanism of Bcl-2 against tumour necrosis factor (TNF)-mediated cell death, the bcl2 gene was transfected into the L929 cells and stably expressed. Two clones having different sensitivity among bcl2-transfected L929 clones had been isolated, and termed clone R1 and R2. It was observed that activation of manganese superoxide dismutase (MnSOD) and suppression of Jun kinase of clone R1 and R2 were correlated with protection from TNF cytotoxicity. Upon treatment with TNF, clone R1 and R2 were more resistant than control L929 cells against TNF cytotoxicity and the protective effect of clone R1 was stronger than clone R2. However, in case of TNF plus actinomycin D treatment, clone R1 was still resistant against TNF cytotoxicity, whereas clone R2 became more sensitive than control L929 cells. The JNK activities of clone R1 and R2 were suppressed upon TNF treatment and in case of TNF plus actinomycin D treatment, clone R2 showed a marked increase in JNK activities and had higher activity than control L929 cells. The specific activities of MnSOD of clone R1 and R2 upon TNF treatment were 70 U/ml and 33 U/ml, respectively, while the MnSOD activity was not detectable in control L929 cells. When TNF and actinomycin D were treated simultaneously, MnSOD activity was not detectable in control L929 cells and bcl2 -transfected L929 cells (clone R1, R2). Consistent with these results, both clone R1 and R2 showed higher levels of MnSOD mRNA expression than control L929 cells after TNF treatment. These data suggest that suppression of Jun kinase and increase of MnSOD may be involved in inhibitory action of Bcl-2 against TNF, and the balance between MnSOD and JNK signalling pathway may be an important factor for the protection of bcl2-transfected L929 cells from TNF cytotoxicity.
Insights
Bcl-2 protein protects cells from tumor necrosis factor (TNF)-mediated death by increasing manganese superoxide dismutase (MnSOD) and decreasing Jun kinase (JNK) activity. This balance is key to cell survival.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Tumor necrosis factor (TNF) is a key mediator of cellular apoptosis.
- Bcl-2 is an anti-apoptotic protein with a known role in cell survival.
- Understanding the precise mechanisms of Bcl-2's protective effects is crucial for cancer therapy.
Purpose of the Study:
- To elucidate the protective mechanism of Bcl-2 against TNF-induced cell death.
- To investigate the role of manganese superoxide dismutase (MnSOD) and Jun kinase (JNK) in Bcl-2-mediated protection.
- To analyze the differential responses of Bcl-2 transfected cells under varying treatment conditions.
Main Methods:
- Transfection of the bcl2 gene into L929 cells to create stable cell lines (clone R1 and R2).
- Treatment of transfected and control cells with TNF and/or actinomycin D.
- Assay of MnSOD activity and JNK activity.
- Measurement of MnSOD mRNA expression levels.
Main Results:
- Bcl-2 transfected clones (R1 and R2) exhibited resistance to TNF-induced cytotoxicity, with clone R1 showing stronger protection.
- TNF treatment led to increased MnSOD activity and mRNA expression, and suppressed JNK activity in both R1 and R2 clones.
- Under combined TNF and actinomycin D treatment, clone R2 became sensitive, showing increased JNK activity, while clone R1 remained resistant.
- MnSOD activity was undetectable when cells were treated with both TNF and actinomycin D.
Conclusions:
- Suppression of JNK signaling and activation of MnSOD are critical components of Bcl-2's inhibition of TNF-mediated cell death.
- The balance between MnSOD and JNK pathways is a significant factor in the cytoprotection conferred by Bcl-2.
- These findings provide insights into novel therapeutic strategies targeting cell death pathways in cancer.