Apoptosis Modulation of Epstein–Barr Virus-Encoded Latent Membrane Protein 1 in the Epithelial Cell Line HeLa Is Stimulus-Dependent

Abstract

Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) is required for viral transformation and has been shown to protect lymphocytes from apoptosis. However, the effect of LMP1 on cells of epithelial origin remains poorly understood. Using the epithelial cell line HeLa in which the expression of LMP1 is inducibly regulated by tetracycline, we demonstrate that apoptosis triggered by ligation of the death receptor, Fas, or by the chemotherapeutic agent, etoposide, is potentiated by LMP1. Apoptosis was assessed by nuclear condensation and activation of caspase-3-like enzymes with concomitant proteolysis of the nuclear caspase substrate, poly(ADP-ribose) polymerase. However, the effect of LMP1 in HeLa cells appeared to be stimulus-dependent since apoptosis induced by tumor necrosis factor (TNF) was inhibited. Moreover, we observed an upregulation of the zinc finger protein A20 and a decrease in expression of Bcl-2 upon induction of LMP1 in HeLa cells. Taken together, these data further our understanding of the function of LMP1 in epithelial cells and suggest that LMP1, similar to its mammalian homolog CD40, can exert opposing effects on cell survival depending on the nature of the apoptosis trigger.

Epstein‐Barr virus‐encoded latent membrane protein 1 promotes stress‐induced apoptosis upstream of caspase‐2‐dependent mitochondrial perturbation

“…4 Stable transfection of TW03 cells was performed by transfecting cells with the LMP1 gene in a mycophenolic acid (MPA) vector, as previously described. 15 Briefly, TW03 cells were seeded in 6-or 12-well culture plates and incubated overnight.…”

“…In addition, determination of NF B-driven luciferase activity was performed as previously described. 4 Briefly, HeLa cells were co-transfected with one of the plasmids above and pCMV ␤Gal and pConA B luc (containing 3 B-binding sites upstream of the luciferase gene). Transfection efficiency was normalized by assessment of ␤-galactosidase activity, and relative light units were calculated as the ratio of luciferase values and OD420 values obtained in the ␤-gal assay.…”

“…4 The following specific antibodies were used: S-12, monoclonal antibody against EBV LMP1; 20 AC-15, monoclonal antibody against human ␤-actin (Sigma); C20, rat polyclonal serum against human caspase-2 (Santa Cruz, Santa Cruz, CA). Horseradish peroxidase (HRP)-linked anti-mouse or rabbit immunoglobulin (Ig) (BioRad, Hercules, CA) were utilized as secondary antibodies, and the membranes were developed using the enhanced chemiluminescence (ECL) kit (Amersham, Uppsala, Sweden) according to the manufacturer's instructions.…”

“…4 Previous studies have implicated caspase-2 as the upstream regulator of cellular stress (e.g., etoposide)-induced apoptosis. 12 To further elucidate the effect of LMP1 on etoposide-triggered cell death, we evaluated the activation of caspase-2 in HeLa cells cultivated in the presence or absence of tetracycline.…”

“…3 We have previously reported a stimulus-dependent modulation of apoptosis by LMP1 in the epithelial cell line, HeLa. 4 LMP1 was thus found to potentiate apoptosis induced by etoposide, a chemotherapeutic agent, whereas tumor necrosis factor (TNF)-␣-induced apoptosis was attenuated. Similarly, LMP1 has been shown to sensitize NPC cell lines to cisplatin, a chemotherapeutic drug commonly used in the treatment of solid tumors.…”