FSMP-13. HYPOXIC REGULATION OF LACTATE DEHYDROGENASE GENES (LDHA/B) IN T98 GLIOBLASTOMA MULTIFORME CELLS

Glioblastoma multiforme (GBM) is the most common primary brain cancer and carries a poor prognosis. GBM cells exhibit extensive metabolic alterations that enhance survival and proliferation in the mixed normoxic-hypoxic tumor microenvironment. Lactate dehydrogenase (LDH) enzymes are critical mediators of the normoxic to hypoxic transition in cells. Two LDH genes (A/B) encode monomers that combine to form five isoenzymes (LDH1-5) with different properties for pyruvate to lactate interconversion. Hypoxic induction of LDHA in all cells appears to occur via HIF-1 mediated transcription. However, little is known about hypoxic regulation of LDHB in cancer. We report on hypoxic regulation of LDHA/B in T98G, a rare cell line that has both normal and neoplastic features. Human T98 GBM cell lines were cultured in a humidified incubator at 37° C and 5% CO2 and were grown in normoxia (21% O2) or hypoxia (95% N2, 5% C02) for 72 hours. Relative expression of LDH isoforms 1-5 was assessed using native gel electrophoresis. Expression of the LDHA and LDHB genes was measured using qRT-PCR. LDHA-dominant isoforms (4/5) were detected in T98G cells subjected to normoxia and hypoxia via gel electrophoresis, however, LDHB-dominant isoforms (1/2) were not. The LDHA/B-equimolar isoform (3) was decreased in T98G cells subjected to hypoxia. LDHA gene expression was over two-fold greater than LDHB in normoxia (p = .00256 by one-tailed Mann-Whitney U test), and over nine-fold greater in hypoxia (p = .00256). LDHA:LDHB expression in hypoxia compared to normoxia was significantly different (p = .00256). LDHA expression increased three-fold in hypoxia (p = .00256), while LDHB expression decreased 0.3-fold in hypoxia (p = .03288). We document LDHB dysregulation in T98G cells as the gene is minimally responsive to oxygen. Therapeutic strategies aimed at promoting LDHB expression may complement inhibition of LDHA and reduce GBM survival in hypoxia.

Mitochondrial and plasma membrane lactate transporter and lactate dehydrogenase isoform expression in breast cancer cell lines

We hypothesized that dysregulation of lactate/pyruvate (monocarboxylate) transporters (MCT) and lactate dehydrogenase (LDH) isoforms contribute to the Warburg effect in cancer. Therefore, we assayed for the expression levels and the localizations of MCT ( 1 , 2 , and 4 ), and LDH (A and B) isoforms in breast cancer cell lines MCF-7 and MDA-MB-231 and compared results with those from a control, untransformed primary breast cell line, HMEC 184. Remarkably, MCT1 is not expressed in MDA-MB-231, but MCT1 is expressed in MCF-7 cells, where its abundance is less than in control HMEC 184 cells. When present in HMEC 184 and MCF-7 cells, MCT1 is localized to the plasma membrane. MCT2 and MCT4 were expressed in all the cell lines studied. MCT4 expression was higher in MDA-MB-231 compared with MCF-7 and HMEC 184 cells, whereas MCT2 abundance was higher in MCF-7 compared with MDA-MB-231 and HMEC 184 cells. Unlike MCT1, MCT2 and MCT4 were localized in mitochondria in addition to the plasma membrane. LDHA and LDHB were expressed in all the cell-lines, but abundances were higher in the two cancer cell lines than in the control cells. MCF-7 cells expressed mainly LDHB, while MDA-MB-231 and control cells expressed mainly LDHA. LDH isoforms were localized in mitochondria in addition to the cytosol. These localization patterns were the same in cancerous and control cell lines. In conclusion, MCT and LDH isoforms have distinct expression patterns in two breast cancer cell lines. These differences may contribute to divergent lactate dynamics and oxidative capacities in these cells, and offer possibilities for targeting cancer cells.