Expression levels of genes involved in the glycolytic pathway.

<p>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.</p

Lactate dehydrogenase activity in BL cell lines and LCLs.

<p>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).</p

Different Mechanisms of Regulation of the Warburg Effect in Lymphoblastoid and Burkitt Lymphoma Cells

<div><p>Background</p><p>The Warburg effect is one of the hallmarks of cancer and rapidly proliferating cells. It is known that the hypoxia-inducible factor 1-alpha (HIF1A) and MYC proteins cooperatively regulate expression of the <i>HK2</i> and <i>PDK1</i> genes, respectively, in the Burkitt lymphoma (BL) cell line P493-6, carrying an inducible <i>MYC</i> gene repression system. However, the mechanism of aerobic glycolysis in BL cells has not yet been fully understood.</p><p>Methods and Findings</p><p>Western blot analysis showed that the HIF1A protein was highly expressed in Epstein–Barr virus (EBV)-positive BL cell lines. Using biochemical assays and quantitative PCR (Q-PCR), we found that—unlike in lymphoblastoid cell lines (LCLs)—the MYC protein was the master regulator of the Warburg effect in these BL cell lines. Inhibition of the transactivation ability of MYC had no influence on aerobic glycolysis in LCLs, but it led to decreased expression of MYC-dependent genes and lactate dehydrogenase A (LDHA) activity in BL cells.</p><p>Conclusions</p><p>Our data suggest that aerobic glycolysis, or the Warburg effect, in BL cells is regulated by MYC expressed at high levels, whereas in LCLs, HIF1A is responsible for this phenomenon.</p></div

Statistical analysis of relative HF1A protein expression.

<p>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).</p

Expression levels of genes involved in glycolysis upon treatment with ACF.

<p>Expression levels of a set of genes (<i>GLUTI</i>, <i>HK</i>, <i>LDHA</i>, <i>MCT4</i>, <i>PDK1</i>, <i>PGK1</i>, and <i>PKM2</i>) 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.</p

Biochemical characteristics of B-cell lines

<p>Biochemical characteristics of B-cell lines</p

Biochemical characteristics of BL cell lines.

<p>The BL cells described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136142#pone.0136142.g003" target="_blank">Fig 3</a> and LCL121028 cells were assayed for the concentrations of l-lactate and pyruvate, and for lactate dehydrogenase (LDHA) catalytic activity by colorimetric assays (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136142#sec005" target="_blank">Materials and methods</a> 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.</p

Influence of ACF on the proliferation of BL cell lines and LCLs.

<p>The percentage of living cells is presented as a function of the time of treatment. LCL121028 cells were quickly killed by ACF.</p

Expression levels of genes involved in glycolysis upon treatment with 10058-F4.

<p>Expression levels of a set of genes (<i>GLUTI</i>, <i>HK</i>, <i>LDHA</i>, <i>MCT4</i>, <i>PDK1</i>, <i>PGK1</i>, and <i>PKM2</i>) 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.</p

Expression level of HIF1A in EBV-infected and mitogen-activated B-cells, and in LCLs.

<p><b>A –</b> Western blotting of HIF1A and its hydroxylated form in LCLs (left panel). The membrane was probed with mouse antibody against HIF1A and the rabbit serum against the hydroxylated form of HIF1A (HIF1A-OH). Notice the absence of HIF1A-OH in LCLs (first lane) and the lack of effect of proteasome inhibition. In contrast, high levels of hydroxylated HIF1A were detected in control MCF7 cells upon proteasome inhibition (right panel). <b>B –</b> Western blotting of whole cell lysates of CD40+IL4-activated B-cells, freshly EBV-infected B-cells and LCLs. Cells were treated with 1 mM NiCl₂, mimicking hypoxia-like conditions. Notice that HIF1A, PHD1 and PHD2 levels in LCLs did not change upon treatment with NiCl₂, in contrast to freshly infected cells. <b>C – </b><i>HIF1A</i> expression in CD40+IL4-activated (ABC), freshly EBV-infected (EBC) B-cells and LCLs (LCL) measured by Q-PCR.</p