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Induction
of Inducible Nitric Oxide Synthase Is an Essential Part of Tumor Necrosis
Factor-[alpha]-Induced Apoptosis in MCF-7 and other Epithelial Tumor Cells
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Claudia Binder,
Matthias Schulz, Wolfgang Hiddemann, and Michael Oellerich |
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Departments
of Haematology/Oncology (CB, MS), and Clinical Chemistry (MO), Georg-August-University,
Göttingen, Med. Klinik III (WH), Klinikum Gro[beta]hadern, Ludwig-Maximilians-University,
München, Germany |
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SUMMARY: TNF-[alpha]-induced
cytotoxicity is mediated by the intracellular "death domain" of the 55-kDa
TNF-[alpha] receptor and has been demonstrated to be coupled with induction
of inducible nitric oxide synthase (iNOS), leading to generation of nitric
oxide radicals (NO|b1). Because it is still widely unknown, to what extent
NO|b1 participates in the execution of TNF-[alpha]-induced apoptosis, NO|b1
production, iNOS expression, and enzyme activity in relation to TNF-[alpha]-induced
apoptotic cell death were investigated in the human breast cancer cell line
MCF-7 and various other malignant cell lines. Incubation with TNF-[alpha]
led to induction of iNOS mRNA and protein as well as enhancement of NOS
activity. Augmented synthesis of NO2, the stable end product
of NO|b1 generation, was significantly correlated with augmented rates of
cell death. Measurement of TNF-[alpha]-triggered production of reactive
oxygen species (ROS) suggested a major role for NO|b1 within the generated
oxygen radicals. Dying cells showed characteristic features of apoptosis.
Addition of cycloheximide (CX) enhanced apoptotic cell death by increasing
iNOS activity. L-Nitro-arginine-methyl-ester (L-NAME), a competitive NOS-inhibitor,
and iNOS antisense oligonucleotides effectively prevented NO2
generation and apoptosis. Evaluation of iNOS expression during TNF-[alpha]-induced
cell death in various malignant cell lines demonstrated iNOS positivity
for all TNF-[alpha]-sensitive cells. The only primarily resistant line was
iNOS negative. In a resistant variant of MCF-7, iNOS mRNA was still detectable;
however, treatment with TNF-[alpha] did not enhance NOS activity. TNF-[alpha]
sensitivity and NO2 production were completely restored by the
addition of CX. Taken together, iNOS induction plays an essential role in
TNF-[alpha]-induced apoptosis of the investigated cell lines. Further studies
are necessary to define the impact of NO|b1 in relation to other specific
effectors of apoptosis such as the caspases. |
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