Induction of inducible nitric oxide synthase by Epstein-Barr virus B95-8-derived LMP1 in Balb/3T3 cells promotes stress-induced cell death and impairs LMP1-mediated transformation.
The latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) causes cellular transformation and activation of several intracellular signaling events. In this report, we show that BLMP1 (encoded by the LMP1 gene derived from the B95-8 strain of EBV) triggers the expression of inducible nitric oxide synthase (iNOS) in Balb/3T3 fibroblasts. Intriguingly, NLMP1, a natural sequence variant of LMP1 identified in EBV-positive nasopharyngeal carcinoma biopsy, does not similarly induce iNOS expression. BLMP1-induced iNOS in Balb/3T3 cells is active to produce nitric oxide (NO), and NO production can be blocked by several iNOS inhibitors. When subjected to environmental stress, Balb/3T3 cells that produce NO lose viability more rapidly than non NO-producing cells. Blockage of NO generation by iNOS inhibitors enhances the viability of NO-producing cells under stress conditions. The activities of caspase-3 and c-Jun N-terminal kinase, two important regulators mediating stress-induced apoptosis, are significantly potentiated following heat shock treatment of BLMP1-expressing/NO-producing cells, compared to parental and NLMP1-expressing cells. Furthermore, treatment with iNOS inhibitor augmented the cloning efficiency (in culture) and tumor growth (in nude mice) of BLMP1-expressing/NO-producing cells. Collectively, the results demonstrate that BLMP1 induces iNOS expression and NO production in Balb/3T3 cells, which leads to the alteration of cell functions, including sensitivity to environmental stress, capability to colonize independent of anchorage and tumorigenicity in nude mice. Our data additionally implicate that the differential iNOS induction potential of the two LMP1 forms may represent the basis of a functional difference between the two LMP1 proteins.
Yu
Jau-Song
JS
Department of Cell and Molecular Biology, Institute of Basic Medical Science, Medical College of Chang Gung University, Tao-Yuan, Taiwan, Republic of China. yusong@mail.cgu.edu.tw
Tsai
Hsing-Chen
HC
Wu
Chih-Ching
CC
Weng
Li-Ping
LP
Li
Hsin-Pai
HP
Chung
Pei-Jung
PJ
Chang
Yu-Sun
YS
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D016923:Cell Death / Q000502:physiology*; D002472:Cell Transformation, Viral; D017931:DNA Primers; D004790:Enzyme Induction; D004854:Herpesvirus 4, Human / Q000502:physiology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009569:Nitric Oxide / Q000096:biosynthesis; D019001:Nitric Oxide Synthase / Q000096:biosynthesis*; D052247:Nitric Oxide Synthase Type II; D018384:Oxidative Stress; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014763:Viral Matrix Proteins / Q000502:physiology*
D017931:DNA Primers; C037662:EBV-associated membrane antigen, Epstein-Barr virus; D014763:Viral Matrix Proteins; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse