NF-kappa B inhibition causes spontaneous apoptosis in Epstein-Barr virus-transformed lymphoblastoid cells

Abstract

Epstein-Barr virus (EBV) transforms B lymphocytes into lymphoblastoid cell lines usurping the Notch and tumor necrosis factor receptor pathways to effect transcription including NF-kappaB activation. To determine whether NF-kappaB activity is essential in the growth and survival of EBV-transformed lymphoblastoid cell lines, a nondegradable IkappaBalpha mutant was expressed under tetracycline regulation. Despite continued Bcl-2 and Bcl-x/L expression, NF-kappaB inhibition induced apoptosis as evidenced by poly(ADP-ribose) polymerase cleavage, nuclear condensation and fragmentation, and hypodiploid DNA content. Both caspase 3 and 8 activation and loss of mitochondrial membrane potential were observed in apoptotic cells. However, caspase inhibition failed to block apoptosis. These experiments indicate that NF-kappaB inhibitors may be useful in the therapy of EBV-induced cellular proliferation.

Figure 1

Growth in Tc⁻ media induced Flag-ΔN-IκBα expression and inhibited NF-κB activity. (A) F-ΔN-IκBα expression was induced within 1 day after Tc withdrawal. Western blot analysis using M5 mAb. Lanes 1–3, extracts from cells grown in Tc⁺ media days 1–3; lanes 4–6, extracts from cells grown in Tc⁻ media, days 1–3. (B) EMSA indicated that F-ΔN-IκBα expression inhibited NF-κB activity. Two specific complexes were formed after incubating the extracts from A with a NF-κB probe from the HIV LTR marked κB-1 and κB-2. Lane 1, 100-fold mutant cold competitor. Lane 2, 100-fold excess wild-type cold competitor. Lanes 3–5, extracts from cells grown in Tc⁺ media, days 1–3; lanes 6–8, extracts from cells grown in Tc⁻ media, days 1–3

Figure 2

NF-κB inhibition down-regulated specific gene expression. (A) FACs analysis of ICAM-1 on cells grown in Tc⁺ (dashed line) or Tc- (solid line) media for 3 days. (B) Western blot analysis of extracts were examined for endogenous IκBα, TRAF1, and p53 expression. Lanes 1, 3, and 5 are extracts from cells grown in Tc⁺ media; lanes 2, 4, and 6 are from cells grown in Tc⁻ media. Lanes 1 and 2, day 1; lanes 3 and 4, day 2; and lanes 5 and 6, day 3. (C) c-Myc and LMP-1 expression was unaffected by inhibition of NF-κB. Lanes are as noted in B.

Figure 3

NF-κB inhibition caused apoptosis. (A) Cell growth was determined by counting on a hemacytometer with the inclusion of trypan blue. (Top) Total cell number—positive and negative for trypan blue. (Bottom) Cell number positive for trypan blue uptake. ⧫, Tc⁺ media; ■, Tc⁻ media. Cultures were initially seeded at 10⁵/ml and counted at 24-h intervals thereafter. (B) Examination of DNA content on day 3 showed the accumulation of hypodiploid (<2 N) cells. After ethanol fixation, cells were stained with propidium iodide and examined by FACS. (Top) Tc⁺ media. (Bottom) Tc⁻ media. (C) Confocal microscopy showing apoptotic bodies of cells in B. (Left) Tc⁺. (Right) Tc⁻.

Figure 4

PARP cleavage occurred before detectable activation of caspase-8 or Bid cleavage. Western blot analysis of extracts for pro-caspase 8, Bid, and PARP. Lanes 1, 3, and 5 are extracts from cells grown in Tc⁺ media. Lanes 2, 4, and 6 are from cells grown in Tc⁻ media. Lanes 1 and 2, day 1; lanes 3 and 4, day 2; and lanes 5 and 6, day 3.

Figure 5

Caspase activation was not required for F-ΔN-IκBα-mediated apoptosis. A total of 50 μM Z.VAD-FMK or 0.1% DMSO, the carrier, were added at day 0. Caspase 3 activity was determined by using the colorimetric substrate DEVD-pNA at Day 3. Cell viability was determined as in Fig. 3. PARP cleavage was determined as in Fig. 4. Lane 1, Tc⁺ media plus DMSO. Lane 2, Tc⁻ media plus DMSO. Lane 3, Tc⁻ media with 50 μM z-VAD.FMK.

Figure 6

NF-κB inhibition reduced A1/Bfl-1 expression, but not Bcl-x/L or Bcl-2 expression. (A) Western blot analysis of extracts for Bcl-x/L and Bcl-2. Lanes 1, 3, and 5 are extracts from cells grown in Tc⁺ media. lanes 2, 4, and 6 are from cells grown in Tc⁻ media. Lanes 1 and 2, day 1; lanes 3 and 4, day 2; and lanes 5 and 6, day 3. (B) Northern blot analysis. Five micrograms of total RNA generated on day 2 was analyzed for Bfl-1 (Left) and glyceraldehyde-3-phosphate dehydrogenase, (Right). Lane 1, Tc⁺ media. Lane 2, Tc⁻ media.

Figure 7

NF-κB inhibition caused a loss of mitochondrial potential but not Cyt c release. (A and B) DiOC6 fluorescence. (A) IB4 F-ΔN-IκBα cells grown in Tc⁺ media, gray line, in Tc⁻ media, black line. (B) BJAB, gray line, and BJAB treated with anti-Fas antibody, black line. (C) Cyt c release from the mitochondria. Western blots of S-100 pellets were analyzed for Cyt c. Lane 1, total cell extracts; lane 2, IB4 F-ΔN-IκBα cells, Tc⁺ media; lane 3, IB4 F-ΔN-IκBα cells, Tc⁻ media; lane 4, BJAB; lane 5, BJAB treated with anti-Fas.