Fig 1.
(A)—Expression levels of HIF1A protein assessed by Western blotting. Membranes were probed with mouse monoclonal antibodies against HIF1A and actin. HIF1A was highly expressed in EBV-positive cell lines with Latency III (LCLs, BL41/B95.8, Mutu III clones 99 & 176, Akuba, BL16, BL18, RAJI) compared with EBV-negative (Ramos, BL41, Mutu clones 9 & 30) and EBV-positive Latency I (Rael, BJAB/B95.8, Mutu I clones 59 & 148) cells; (B)—Relative HIF1A protein expression levels, normalized to the actin signal. The ratio of HIF1A to actin signals was calculated using the Western blotting results (Fig 1A). Note the higher expression of HIF1A in EBV-positive cells.
Fig 2.
Statistical analysis of relative HF1A protein expression.
Kruskal–Wallis tests were applied to four groups, comprising seven EBV-negative, six Latency I, seven Latency III BL cell lines, and four LCLs. HIF1A was expressed significantly higher in Latency III cells (p = 0.008<0.05).
Fig 3.
Expression levels of genes involved in the glycolytic pathway.
We studied isogenic BL41 (Bl41, EBV negative; BL41/B95.8, Latency III) and Mutu (Mutu, EBV negative; Mutu I, Latency I; and Mutu III, Latency III) BL cell lines, and LCL121028 cells (5 months old). No significant differences in expression levels were observed in the five groups of cells for the studied genes. Each point represents the median value for three Q-PCR reactions. No value differed by more than 30% of the means.
Table 1.
Biochemical characteristics of B-cell lines
Fig 4.
Biochemical characteristics of BL cell lines.
The BL cells described in Fig 3 and LCL121028 cells were assayed for the concentrations of l-lactate and pyruvate, and for lactate dehydrogenase (LDHA) catalytic activity by colorimetric assays (see Materials and methods section). Latency III BL cells showed significantly higher levels of pyruvate production and LDHA activity (p = 0.0091 and p = 0.0349, respectively). There were no differences in LDHA expression at mRNA levels (no value differed more than 30% from the means). Lactate concentrations were similar in all five studied groups.
Fig 5.
Influence of ACF on the proliferation of BL cell lines and LCLs.
The percentage of living cells is presented as a function of the time of treatment. LCL121028 cells were quickly killed by ACF.
Fig 6.
Expression levels of genes involved in glycolysis upon treatment with ACF.
Expression levels of a set of genes (GLUTI, HK, LDHA, MCT4, PDK1, PGK1, and PKM2) were assessed by Q-PCR after the treatment of cells with 5 μM of ACF for 3 h. Kruskal–Wallis tests were applied to the results for 12 groups; namely, two EBV-negative, one Latency I, two Latency III BL cell lines, and LCL121028 cells—controls and treated with ACF. Note the significant decrease in gene expression under ACF treatment in LCLs (p = 0.0082; framed in the Fig). The median value for three Q-PCR reactions is shown; the standard deviation did not exceed 30% of the means.
Fig 7.
Expression levels of genes involved in glycolysis upon treatment with 10058-F4.
Expression levels of a set of genes (GLUTI, HK, LDHA, MCT4, PDK1, PGK1, and PKM2) were assessed by Q-PCR after the treatment of cells with 100 μM of 10058-F4 for 4 h. Kruskal–Wallis tests were applied to the results for 12 groups: two EBV-negative, one Latency I, and two Latency III BL cell lines, and LCL121028 cells—controls and treated with 10058-F4. Note the significant decreases in gene expression levels under 10058-F4 treatment in the BL cell lines (p = 0.011), in contrast with no change in LCL121028 cells (encircled). The median value for three Q-PCR reactions is shown; the standard deviation did not exceed 30% of the means.
Fig 8.
Lactate dehydrogenase activity in BL cell lines and LCLs.
LDHA catalytic activity was measured in the control cells and after treatment with 100 μM of 10058-F4 for 4 h. The median value of three measurements (standard deviation not exceeding 30%) is shown on the Fig. Each of the four groups consisted of three cell lines: EBV-negative (Ramos, Bl28, DG75), Latency I (BL28/B95.8, Rael, Akata (+)), and Latency III (BL16, BL18, RAJI) BL cell lines, and three LCLs (051128, 111210, and 120214). Kruskal–Wallis tests were applied to the results of eight groups (controls and those treated with 10058-F4). Note the significant decrease of LDHA activity in all the BL lines upon inhibition of MYC transactivation ability (p = 0.0118). The catalytic activity of LDHA was not changed in LCLs (encircled).