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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) NKL and BJAB were infected with Akata-derived EBV. Southern blot analysis detected monoclonal EBV genome in EBV-infected NKL cells. The blotting signal for clone 2 (lane 4) is weaker than those of established EBV-infected NKL sublines named TL1 (lane 5) and TL2 (lane 6), suggesting that clone 2 should contain less copy number of EBV. EBV-infected BJAB appears to consist of two clones (lane 2). (B) TL1 (lane 5) and TL2 (lane 6) express EBNA1 and LMP1, but lack EBNA2 and lytic marker proteins, ZEBRA and EA-D. TPA-treated Akata (lane 1) and EBV-infected BJAB (lane 2) are positive controls of lytic and latent phases, respectively. (C) Time courses of cell count for NKL, TL1, and TL2 at steady state. TL1 and TL2 showed no growth advantage compared with NKL. Alive (trypan-blue negative)- and dead (trypan-blue positive)-cell counts are shown in gray- and black-bars, respectively. (D) Time courses of cell count for NKL, TL1, and TL2 after treatment with 150 nM of doxorubicin (DXR). After 48 h, approximately 60% of NKL cells were dead, whereas above 85% of TL1 and TL2 cells survived. (E) Change in cell morphology after 48 h of treatment with DXR in three lines (Wright–Giemsa stain × 1000). Although both TL1 and TL2 cells had no apparent change in their cell morphology, about half of NKL cells underwent apoptosis (arrowheads). (F) A cell viability assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide after 48 h of treatment with 4-hydroxycyclophosphamide, DXR, vincristine, and BMS-345541, TL1 and TL2 showed resistance to DXR and VCR treatment compared with NKL. (G) Flow cytometric terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay after treatment with 150 nM of DXR or serum depletion (from 10 to 0.1% of fetal bovine serum). Although about half of NKL cells underwent apoptosis after each treatment, essentially no apoptotic populations were detected in TL1 and TL2. Open arrowheads show TUNEL-positive populations. (H) 7-amino actinomycin D rejection and annexin V-binding assay after treatment with DXR or serum depletion. About half of NKL cells became positive for annexin V after each treatment. In contrast, approximately 90% of TL1 and TL2 cells were negative for annexin V after each treatment.
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fig1: (A) NKL and BJAB were infected with Akata-derived EBV. Southern blot analysis detected monoclonal EBV genome in EBV-infected NKL cells. The blotting signal for clone 2 (lane 4) is weaker than those of established EBV-infected NKL sublines named TL1 (lane 5) and TL2 (lane 6), suggesting that clone 2 should contain less copy number of EBV. EBV-infected BJAB appears to consist of two clones (lane 2). (B) TL1 (lane 5) and TL2 (lane 6) express EBNA1 and LMP1, but lack EBNA2 and lytic marker proteins, ZEBRA and EA-D. TPA-treated Akata (lane 1) and EBV-infected BJAB (lane 2) are positive controls of lytic and latent phases, respectively. (C) Time courses of cell count for NKL, TL1, and TL2 at steady state. TL1 and TL2 showed no growth advantage compared with NKL. Alive (trypan-blue negative)- and dead (trypan-blue positive)-cell counts are shown in gray- and black-bars, respectively. (D) Time courses of cell count for NKL, TL1, and TL2 after treatment with 150 nM of doxorubicin (DXR). After 48 h, approximately 60% of NKL cells were dead, whereas above 85% of TL1 and TL2 cells survived. (E) Change in cell morphology after 48 h of treatment with DXR in three lines (Wright–Giemsa stain × 1000). Although both TL1 and TL2 cells had no apparent change in their cell morphology, about half of NKL cells underwent apoptosis (arrowheads). (F) A cell viability assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide after 48 h of treatment with 4-hydroxycyclophosphamide, DXR, vincristine, and BMS-345541, TL1 and TL2 showed resistance to DXR and VCR treatment compared with NKL. (G) Flow cytometric terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay after treatment with 150 nM of DXR or serum depletion (from 10 to 0.1% of fetal bovine serum). Although about half of NKL cells underwent apoptosis after each treatment, essentially no apoptotic populations were detected in TL1 and TL2. Open arrowheads show TUNEL-positive populations. (H) 7-amino actinomycin D rejection and annexin V-binding assay after treatment with DXR or serum depletion. About half of NKL cells became positive for annexin V after each treatment. In contrast, approximately 90% of TL1 and TL2 cells were negative for annexin V after each treatment.

Mentions: We initially obtained three G418-resistant NKL subclones containing monoclonal EBV genome (Figure 1A). During the 4 years of incubation, clone 2 stopped proliferation, and we finally established two EBV-infected sublines named TL1 and TL2. Western blot analysis showed expression of EBNA1 in all EBV-infected subclones (Figure 1B). EBNA2 and lytic marker proteins, ZEBRA and EA-D were absent in these clones. In addition, LMP1 was detected in TL1 and TL2 but not in clone 2. LMP1 in TL1 and TL2 showed smaller sizes than that in EBV-infected BJAB (Figure 1B). These truncated forms resemble lytic LMP1 detected in TPA-treated Akata (Figure 1B). Because lytic marker proteins were absent in both sublines, detection of the truncated forms may represent rapid turnover of LMP1 in TL1 and TL2.


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) NKL and BJAB were infected with Akata-derived EBV. Southern blot analysis detected monoclonal EBV genome in EBV-infected NKL cells. The blotting signal for clone 2 (lane 4) is weaker than those of established EBV-infected NKL sublines named TL1 (lane 5) and TL2 (lane 6), suggesting that clone 2 should contain less copy number of EBV. EBV-infected BJAB appears to consist of two clones (lane 2). (B) TL1 (lane 5) and TL2 (lane 6) express EBNA1 and LMP1, but lack EBNA2 and lytic marker proteins, ZEBRA and EA-D. TPA-treated Akata (lane 1) and EBV-infected BJAB (lane 2) are positive controls of lytic and latent phases, respectively. (C) Time courses of cell count for NKL, TL1, and TL2 at steady state. TL1 and TL2 showed no growth advantage compared with NKL. Alive (trypan-blue negative)- and dead (trypan-blue positive)-cell counts are shown in gray- and black-bars, respectively. (D) Time courses of cell count for NKL, TL1, and TL2 after treatment with 150 nM of doxorubicin (DXR). After 48 h, approximately 60% of NKL cells were dead, whereas above 85% of TL1 and TL2 cells survived. (E) Change in cell morphology after 48 h of treatment with DXR in three lines (Wright–Giemsa stain × 1000). Although both TL1 and TL2 cells had no apparent change in their cell morphology, about half of NKL cells underwent apoptosis (arrowheads). (F) A cell viability assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide after 48 h of treatment with 4-hydroxycyclophosphamide, DXR, vincristine, and BMS-345541, TL1 and TL2 showed resistance to DXR and VCR treatment compared with NKL. (G) Flow cytometric terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay after treatment with 150 nM of DXR or serum depletion (from 10 to 0.1% of fetal bovine serum). Although about half of NKL cells underwent apoptosis after each treatment, essentially no apoptotic populations were detected in TL1 and TL2. Open arrowheads show TUNEL-positive populations. (H) 7-amino actinomycin D rejection and annexin V-binding assay after treatment with DXR or serum depletion. About half of NKL cells became positive for annexin V after each treatment. In contrast, approximately 90% of TL1 and TL2 cells were negative for annexin V after each treatment.
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fig1: (A) NKL and BJAB were infected with Akata-derived EBV. Southern blot analysis detected monoclonal EBV genome in EBV-infected NKL cells. The blotting signal for clone 2 (lane 4) is weaker than those of established EBV-infected NKL sublines named TL1 (lane 5) and TL2 (lane 6), suggesting that clone 2 should contain less copy number of EBV. EBV-infected BJAB appears to consist of two clones (lane 2). (B) TL1 (lane 5) and TL2 (lane 6) express EBNA1 and LMP1, but lack EBNA2 and lytic marker proteins, ZEBRA and EA-D. TPA-treated Akata (lane 1) and EBV-infected BJAB (lane 2) are positive controls of lytic and latent phases, respectively. (C) Time courses of cell count for NKL, TL1, and TL2 at steady state. TL1 and TL2 showed no growth advantage compared with NKL. Alive (trypan-blue negative)- and dead (trypan-blue positive)-cell counts are shown in gray- and black-bars, respectively. (D) Time courses of cell count for NKL, TL1, and TL2 after treatment with 150 nM of doxorubicin (DXR). After 48 h, approximately 60% of NKL cells were dead, whereas above 85% of TL1 and TL2 cells survived. (E) Change in cell morphology after 48 h of treatment with DXR in three lines (Wright–Giemsa stain × 1000). Although both TL1 and TL2 cells had no apparent change in their cell morphology, about half of NKL cells underwent apoptosis (arrowheads). (F) A cell viability assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide after 48 h of treatment with 4-hydroxycyclophosphamide, DXR, vincristine, and BMS-345541, TL1 and TL2 showed resistance to DXR and VCR treatment compared with NKL. (G) Flow cytometric terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay after treatment with 150 nM of DXR or serum depletion (from 10 to 0.1% of fetal bovine serum). Although about half of NKL cells underwent apoptosis after each treatment, essentially no apoptotic populations were detected in TL1 and TL2. Open arrowheads show TUNEL-positive populations. (H) 7-amino actinomycin D rejection and annexin V-binding assay after treatment with DXR or serum depletion. About half of NKL cells became positive for annexin V after each treatment. In contrast, approximately 90% of TL1 and TL2 cells were negative for annexin V after each treatment.
Mentions: We initially obtained three G418-resistant NKL subclones containing monoclonal EBV genome (Figure 1A). During the 4 years of incubation, clone 2 stopped proliferation, and we finally established two EBV-infected sublines named TL1 and TL2. Western blot analysis showed expression of EBNA1 in all EBV-infected subclones (Figure 1B). EBNA2 and lytic marker proteins, ZEBRA and EA-D were absent in these clones. In addition, LMP1 was detected in TL1 and TL2 but not in clone 2. LMP1 in TL1 and TL2 showed smaller sizes than that in EBV-infected BJAB (Figure 1B). These truncated forms resemble lytic LMP1 detected in TPA-treated Akata (Figure 1B). Because lytic marker proteins were absent in both sublines, detection of the truncated forms may represent rapid turnover of LMP1 in TL1 and TL2.

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