Figure 1.
Figure 1. Increased growth and survival of EBV-positive HL cells expressing a limited repertoire of EBV genes is associated with the transcriptional up-regulation of autotaxin. (A) RT-PCR analysis demonstrates a pattern of virus gene expression in EBV-infected KM-H2 (KM-H2-Akata) cells predominated by expression of the EBERs, Qp-driven EBNA1, and the BamH1A transcripts. There was also very low-level transcription from Cp/Wp and associated low levels of EBNA2 and LMP2 transcripts in these cells. Neither LMP1 RNA nor protein (data not shown) could be detected in these cells. (B) Loss of EBER expression in 2 clones (SD3 and SD5) of EBV-negative L591 cells derived from the EBV-positive parental line by serial dilution. Quantitative PCR was also used to confirm the loss of the EBV genome from these cells (data not shown). Clone SD3 was used in later experiments. (C) (upper panels) WST1 assays showing relative metabolic activity (cell growth) of control cells (KMH2-neo) adjusted to a value of 1 compared with EBV-infected KM-H2 cells (KM-H2 + Akata) in 10% and 1% sera. A clear and significant difference in cell growth between control and EBV-infected cells is observed. (lower panels) Cell viability assays at 10% and 0.1% serum, demonstrating consistent and significant increases in the viability of EBV-infected KM-H2 cells compared with controls. ▪ indicates KM-H2-neo; and □, KM-H2 + Akata. (D) L591 SD3 cells showed dramatically reduced viability compared with EBV-positive parental L591 cells in 10% and 1% sera. Cell proliferation was similarly affected by EBV loss (data not shown). ▪ indicates L591; and □, L591-SD3. (E) Heat map showing gene expression differences across 4 replicates of EBV-negative KM-H2 cells and EBV-infected KM-H2 cells (KM-H2 Akata); 26 probe sets met the criteria of a positive or negative change of 2.5-fold or greater and a false discovery rate of 5% or less. Eleven probe sets were up-regulated and 15 were down-regulated after EBV infection. The most highly up-regulated probe set (mean fold increase, 4.16) was autotaxin (ENPP2). (F) Confirmation of increased autotaxin mRNA in EBV-infected KM-H2 (KM-H2 Akata) and L591 cells by RT-PCR analysis compared with EBV-negative parental KM-H2, KM-H2 neo control, and L591-SD3 cells. Other EBV-negative HL cell lines (L428, L540, L1236) and the EBV-transformed lymphoblastoid cell line X50-7 expressed lower levels of autotaxin mRNA.

Increased growth and survival of EBV-positive HL cells expressing a limited repertoire of EBV genes is associated with the transcriptional up-regulation of autotaxin. (A) RT-PCR analysis demonstrates a pattern of virus gene expression in EBV-infected KM-H2 (KM-H2-Akata) cells predominated by expression of the EBERs, Qp-driven EBNA1, and the BamH1A transcripts. There was also very low-level transcription from Cp/Wp and associated low levels of EBNA2 and LMP2 transcripts in these cells. Neither LMP1 RNA nor protein (data not shown) could be detected in these cells. (B) Loss of EBER expression in 2 clones (SD3 and SD5) of EBV-negative L591 cells derived from the EBV-positive parental line by serial dilution. Quantitative PCR was also used to confirm the loss of the EBV genome from these cells (data not shown). Clone SD3 was used in later experiments. (C) (upper panels) WST1 assays showing relative metabolic activity (cell growth) of control cells (KMH2-neo) adjusted to a value of 1 compared with EBV-infected KM-H2 cells (KM-H2 + Akata) in 10% and 1% sera. A clear and significant difference in cell growth between control and EBV-infected cells is observed. (lower panels) Cell viability assays at 10% and 0.1% serum, demonstrating consistent and significant increases in the viability of EBV-infected KM-H2 cells compared with controls. ▪ indicates KM-H2-neo; and □, KM-H2 + Akata. (D) L591 SD3 cells showed dramatically reduced viability compared with EBV-positive parental L591 cells in 10% and 1% sera. Cell proliferation was similarly affected by EBV loss (data not shown). ▪ indicates L591; and □, L591-SD3. (E) Heat map showing gene expression differences across 4 replicates of EBV-negative KM-H2 cells and EBV-infected KM-H2 cells (KM-H2 Akata); 26 probe sets met the criteria of a positive or negative change of 2.5-fold or greater and a false discovery rate of 5% or less. Eleven probe sets were up-regulated and 15 were down-regulated after EBV infection. The most highly up-regulated probe set (mean fold increase, 4.16) was autotaxin (ENPP2). (F) Confirmation of increased autotaxin mRNA in EBV-infected KM-H2 (KM-H2 Akata) and L591 cells by RT-PCR analysis compared with EBV-negative parental KM-H2, KM-H2 neo control, and L591-SD3 cells. Other EBV-negative HL cell lines (L428, L540, L1236) and the EBV-transformed lymphoblastoid cell line X50-7 expressed lower levels of autotaxin mRNA.

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