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Epstein-Barr virus renders the infected natural killer cell line, NKL resistant to doxorubicin-induced apoptosis.

Isobe Y, Sugimoto K, Matsuura I, Takada K, Oshimi K - Br. J. Cancer (2008)

Bottom Line: We established two Epstein-Barr virus (EBV)-infected NKL sublines, which acquired stress resistant phenotype against DNA damage and starvation compared with EBV-negative NKL.EBV-rendered doxorubicin resistance at least partially through NF-kappaB activation and the resultant sustenance of antiapoptotic proteins including Bcl-X(L) and FLIP(L/S).

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology, Juntendo University School of Medicine, Bunkyo-ku, Tokyo 113-8421, Japan.

ABSTRACT
We established two Epstein-Barr virus (EBV)-infected NKL sublines, which acquired stress resistant phenotype against DNA damage and starvation compared with EBV-negative NKL. EBV-rendered doxorubicin resistance at least partially through NF-kappaB activation and the resultant sustenance of antiapoptotic proteins including Bcl-X(L) and FLIP(L/S).

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(A) Western blot analysis showed essentially the same expression levels of Bcl-2, Bcl-XL, Mcl-1, Bax, FLIPL/S, and p53 among NKL, TL1 and TL2. (B) Cell-surface P-glycoprotein was absent not only in NKL but also TL1 and TL2 by flow cytometry. Open histograms show negative control with isotype-matched control antibody. (C) Alterations of antiapoptotic protein levels in NKL and two sublines after doxorubicin (DXR) treatment. Although NKL declined expression levels of Bcl-2, Bcl-XL, and FLIPL/S after 24 h of DXR treatment (left column), TL1 (middle column) and TL2 (right column) kept to express these proteins, and showed rather increased expression levels of Bcl-XL and FLIPL/S. (D) NF-κB (p65 subunit) binding activity after DXR treatment. Although the binding activity in NKL remained to be its basal level at 24 h, those in TL1 and TL2 increased approximately five times after 12 h and further boosted at 24 h. In contrast, NF-κB inhibitor BMS-345541 almost completely suppressed DXR-induced NF-κB activation in all three lines. Each experiment was performed in triplicate. Each error bar represents s.d. (E) Alterations of antiapoptotic protein levels in NKL and two sublines after treatment with both DXR and BMS-345541. In the presence of BMS-345541, treatment with DXR clearly decreased expression levels of Bcl-XL and FLIPL/S even in TL1 and TL2. Expression level of Bcl-2 was decreased only in NKL. (F) 7-amino actinomycin D rejection and annexin V-binding assay after treatment with DMSO (control), BMS-345541, DXR, and both BMS-345541 and DXR. Although approximately 5–7% of apoptotic populations were detected in TL1 and TL2 after treatment with 3 μM of BMS-345541 and 150 nM of DXR, respectively, those mixed treatments induced apoptosis in approximately 40% of populations in TL1 and TL2.
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fig2: (A) Western blot analysis showed essentially the same expression levels of Bcl-2, Bcl-XL, Mcl-1, Bax, FLIPL/S, and p53 among NKL, TL1 and TL2. (B) Cell-surface P-glycoprotein was absent not only in NKL but also TL1 and TL2 by flow cytometry. Open histograms show negative control with isotype-matched control antibody. (C) Alterations of antiapoptotic protein levels in NKL and two sublines after doxorubicin (DXR) treatment. Although NKL declined expression levels of Bcl-2, Bcl-XL, and FLIPL/S after 24 h of DXR treatment (left column), TL1 (middle column) and TL2 (right column) kept to express these proteins, and showed rather increased expression levels of Bcl-XL and FLIPL/S. (D) NF-κB (p65 subunit) binding activity after DXR treatment. Although the binding activity in NKL remained to be its basal level at 24 h, those in TL1 and TL2 increased approximately five times after 12 h and further boosted at 24 h. In contrast, NF-κB inhibitor BMS-345541 almost completely suppressed DXR-induced NF-κB activation in all three lines. Each experiment was performed in triplicate. Each error bar represents s.d. (E) Alterations of antiapoptotic protein levels in NKL and two sublines after treatment with both DXR and BMS-345541. In the presence of BMS-345541, treatment with DXR clearly decreased expression levels of Bcl-XL and FLIPL/S even in TL1 and TL2. Expression level of Bcl-2 was decreased only in NKL. (F) 7-amino actinomycin D rejection and annexin V-binding assay after treatment with DMSO (control), BMS-345541, DXR, and both BMS-345541 and DXR. Although approximately 5–7% of apoptotic populations were detected in TL1 and TL2 after treatment with 3 μM of BMS-345541 and 150 nM of DXR, respectively, those mixed treatments induced apoptosis in approximately 40% of populations in TL1 and TL2.

Mentions: We next evaluated possible alterations of antiapoptotic proteins after EBV infection of NKL cells. Western blot analysis showed essentially the same levels of Bcl-2, Bcl-XL, Mcl-1, Bax, p53 and FLIPL/S among NKL, TL1 and TL2 (Figure 2A). Expression of MDR1-encoded P-gp was absent in all these lines, which confirmed that the resistance to DXR should be unrelated to drug pumping (Figure 2B). Although no apparent difference was detected at the steady state, treatment with 150 nM of DXR for 24 h reduced expression levels of Bcl-2, Bcl-XL, and FLIPL/S specifically in NKL (Figure 2C). The amounts of these proteins were constant or rather increased in TL1 and TL2 after the same treatment. Protein levels of Mcl-1 and Bax were unchanged in all three lines (Figure 2C). Increased levels of p53 phosphorylation at serine-15 after DXR treatment indicated preservation of normal p53 activity in all three lines (Figure 2C). Because nuclear factor (NF)-κB is reported to affect expression levels of Bcl-XL and FLIPL/S (Karin, 2006), we examined possible involvement of NF-κB in the EBV-mediated resistance to DXR. In spite of a transient increase at 12 h, target sequences-binding activity of NF-κB p65 clearly regressed after 24 h of DXR treatment in NKL (Figure 2D). In contrast, although the basal activities in TL1 and TL2 were rather suppressed, they increased approximately five times as high as their basal levels after 12 h of DXR treatment, and were further boosted at 24 h (Figure 2D). However, NF-κB inhibitor BMS-345541 almost completely suppressed DXR-induced NF-κB activation in all three lines. In the presence of this inhibitor, treatment with DXR clearly decreased expression levels of Bcl-XL and FLIPL/S and induced massive apoptosis even in TL1 and TL2 (Figure 2E and F). It should be noted that BMS-345541 rather decreased the apoptotic cell percentage from approximately 60% to around 35% in DXR-treated NKL. These experiments were repeated at least three times with essentially the same results.


Epstein-Barr virus renders the infected natural killer cell line, NKL resistant to doxorubicin-induced apoptosis.

Isobe Y, Sugimoto K, Matsuura I, Takada K, Oshimi K - Br. J. Cancer (2008)

(A) Western blot analysis showed essentially the same expression levels of Bcl-2, Bcl-XL, Mcl-1, Bax, FLIPL/S, and p53 among NKL, TL1 and TL2. (B) Cell-surface P-glycoprotein was absent not only in NKL but also TL1 and TL2 by flow cytometry. Open histograms show negative control with isotype-matched control antibody. (C) Alterations of antiapoptotic protein levels in NKL and two sublines after doxorubicin (DXR) treatment. Although NKL declined expression levels of Bcl-2, Bcl-XL, and FLIPL/S after 24 h of DXR treatment (left column), TL1 (middle column) and TL2 (right column) kept to express these proteins, and showed rather increased expression levels of Bcl-XL and FLIPL/S. (D) NF-κB (p65 subunit) binding activity after DXR treatment. Although the binding activity in NKL remained to be its basal level at 24 h, those in TL1 and TL2 increased approximately five times after 12 h and further boosted at 24 h. In contrast, NF-κB inhibitor BMS-345541 almost completely suppressed DXR-induced NF-κB activation in all three lines. Each experiment was performed in triplicate. Each error bar represents s.d. (E) Alterations of antiapoptotic protein levels in NKL and two sublines after treatment with both DXR and BMS-345541. In the presence of BMS-345541, treatment with DXR clearly decreased expression levels of Bcl-XL and FLIPL/S even in TL1 and TL2. Expression level of Bcl-2 was decreased only in NKL. (F) 7-amino actinomycin D rejection and annexin V-binding assay after treatment with DMSO (control), BMS-345541, DXR, and both BMS-345541 and DXR. Although approximately 5–7% of apoptotic populations were detected in TL1 and TL2 after treatment with 3 μM of BMS-345541 and 150 nM of DXR, respectively, those mixed treatments induced apoptosis in approximately 40% of populations in TL1 and TL2.
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Related In: Results  -  Collection

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fig2: (A) Western blot analysis showed essentially the same expression levels of Bcl-2, Bcl-XL, Mcl-1, Bax, FLIPL/S, and p53 among NKL, TL1 and TL2. (B) Cell-surface P-glycoprotein was absent not only in NKL but also TL1 and TL2 by flow cytometry. Open histograms show negative control with isotype-matched control antibody. (C) Alterations of antiapoptotic protein levels in NKL and two sublines after doxorubicin (DXR) treatment. Although NKL declined expression levels of Bcl-2, Bcl-XL, and FLIPL/S after 24 h of DXR treatment (left column), TL1 (middle column) and TL2 (right column) kept to express these proteins, and showed rather increased expression levels of Bcl-XL and FLIPL/S. (D) NF-κB (p65 subunit) binding activity after DXR treatment. Although the binding activity in NKL remained to be its basal level at 24 h, those in TL1 and TL2 increased approximately five times after 12 h and further boosted at 24 h. In contrast, NF-κB inhibitor BMS-345541 almost completely suppressed DXR-induced NF-κB activation in all three lines. Each experiment was performed in triplicate. Each error bar represents s.d. (E) Alterations of antiapoptotic protein levels in NKL and two sublines after treatment with both DXR and BMS-345541. In the presence of BMS-345541, treatment with DXR clearly decreased expression levels of Bcl-XL and FLIPL/S even in TL1 and TL2. Expression level of Bcl-2 was decreased only in NKL. (F) 7-amino actinomycin D rejection and annexin V-binding assay after treatment with DMSO (control), BMS-345541, DXR, and both BMS-345541 and DXR. Although approximately 5–7% of apoptotic populations were detected in TL1 and TL2 after treatment with 3 μM of BMS-345541 and 150 nM of DXR, respectively, those mixed treatments induced apoptosis in approximately 40% of populations in TL1 and TL2.
Mentions: We next evaluated possible alterations of antiapoptotic proteins after EBV infection of NKL cells. Western blot analysis showed essentially the same levels of Bcl-2, Bcl-XL, Mcl-1, Bax, p53 and FLIPL/S among NKL, TL1 and TL2 (Figure 2A). Expression of MDR1-encoded P-gp was absent in all these lines, which confirmed that the resistance to DXR should be unrelated to drug pumping (Figure 2B). Although no apparent difference was detected at the steady state, treatment with 150 nM of DXR for 24 h reduced expression levels of Bcl-2, Bcl-XL, and FLIPL/S specifically in NKL (Figure 2C). The amounts of these proteins were constant or rather increased in TL1 and TL2 after the same treatment. Protein levels of Mcl-1 and Bax were unchanged in all three lines (Figure 2C). Increased levels of p53 phosphorylation at serine-15 after DXR treatment indicated preservation of normal p53 activity in all three lines (Figure 2C). Because nuclear factor (NF)-κB is reported to affect expression levels of Bcl-XL and FLIPL/S (Karin, 2006), we examined possible involvement of NF-κB in the EBV-mediated resistance to DXR. In spite of a transient increase at 12 h, target sequences-binding activity of NF-κB p65 clearly regressed after 24 h of DXR treatment in NKL (Figure 2D). In contrast, although the basal activities in TL1 and TL2 were rather suppressed, they increased approximately five times as high as their basal levels after 12 h of DXR treatment, and were further boosted at 24 h (Figure 2D). However, NF-κB inhibitor BMS-345541 almost completely suppressed DXR-induced NF-κB activation in all three lines. In the presence of this inhibitor, treatment with DXR clearly decreased expression levels of Bcl-XL and FLIPL/S and induced massive apoptosis even in TL1 and TL2 (Figure 2E and F). It should be noted that BMS-345541 rather decreased the apoptotic cell percentage from approximately 60% to around 35% in DXR-treated NKL. These experiments were repeated at least three times with essentially the same results.

Bottom Line: We established two Epstein-Barr virus (EBV)-infected NKL sublines, which acquired stress resistant phenotype against DNA damage and starvation compared with EBV-negative NKL.EBV-rendered doxorubicin resistance at least partially through NF-kappaB activation and the resultant sustenance of antiapoptotic proteins including Bcl-X(L) and FLIP(L/S).

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology, Juntendo University School of Medicine, Bunkyo-ku, Tokyo 113-8421, Japan.

ABSTRACT
We established two Epstein-Barr virus (EBV)-infected NKL sublines, which acquired stress resistant phenotype against DNA damage and starvation compared with EBV-negative NKL. EBV-rendered doxorubicin resistance at least partially through NF-kappaB activation and the resultant sustenance of antiapoptotic proteins including Bcl-X(L) and FLIP(L/S).

Show MeSH
Related in: MedlinePlus