Expression of TNF-alpha leader sequence renders MCF-7 tumor cells resistant to the cytotoxicity of soluble TNF-alpha.
Transmembrane TNF-alpha (tmTNF-alpha) contains a leader sequence (LS) that can be phosphorylated and cleaved at its cytoplasmic portion, inducing IL-12 production. We observed that the breast cancer cell line MDA-MB-231 expressing transmembrane TNF-alpha (tmTNF-alpha) at high level was resistant to soluble TNF-alpha (sTNF-alpha)-induced cytotoxicity, accompanied by constitutive NF-kappaB activation. In contrast, MCF-7 cells expressing tmTNF-alpha at very low level were sensitive to sTNF-alpha-induced cell death and had no detectable NF-kappaB activation. Consistently, siRNA-mediated tmTNF-alpha knockdown blocked NF-kappaB activation and rendered MDA-MB-231 sensitive. To test our hypothesis that TNF-LS may play an important role in determining the sensitivity of tumor cells to sTNF-alpha, we stably transfected MCF-7 cells with TNF-LS. We found that transfection of TNF-LS or wild-type TNF-alpha containing LS constitutively activated NF-kappaB and conferred the cytotoxic resistance of MCF-7 cells, while transfection of a mutant tmTNF-alpha lacking the cytoplasmic segment of LS neither activated NF-kappaB nor affected the sensitivity. However, NF-kappaB inhibitor PDTC suppressed NF-kappaB activation and reconstituted sensitivity of TNF-LS/MCF-7 cells. To check whether TNF-LS is required to be cleaved or internalized for NF-kappaB activation to occur, we used signal peptide peptidase inhibitor (Z-LL)(2)-ketone and receptor internalization inhibitor MDC to treat cells. Interestingly, both inhibitors increased TNF-LS expression on the cell surface and enhanced NF-kappaB activation. These results indicate that membrane-anchored TNF-LS contributes to constitutive activation of NF-kappaB and resistance to sTNF-alpha-induced cell death. Therefore, TNF-LS appears to be responsible for tmTNF-alpha-induced resistance in the breast cancer cells.
Yan
Dan
D
Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, People's Republic of China.
Qin
Nalin
N
Zhang
Hailong
H
Liu
Tao
T
Yu
Mingxia
M
Jiang
Xiaodan
X
Feng
Wei
W
Wang
Jing
J
Yin
Bingjiao
B
Zhang
Tao
T
Zhou
Muxiang
M
Li
Zhuoya
Z
D015153:Blotting, Western; D001943:Breast Neoplasms / Q000378:metabolism*; D016923:Cell Death / Q000502:physiology; D045744:Cell Line, Tumor; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005455:Fluorescent Antibody Technique; D006801:Humans; D018613:Microscopy, Confocal; D016328:NF-kappa B / Q000378:metabolism; D021382:Protein Sorting Signals / Q000502:physiology*; D034741:RNA, Small Interfering; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D016328:NF-kappa B; D021382:Protein Sorting Signals; D034741:RNA, Small Interfering; D014409:Tumor Necrosis Factor-alpha