Pediatric glioblastoma cells are sensitive to drugs that inhibit eIF2α dephosphorylation and its phosphomimetic S51D variant.

We found that pediatric glioblastoma (PED-GBM) cell lines from diffuse intrinsic pontine glioma (DIPG) carrying the H3K27M mutation or from diffuse hemispheric glioma expressing the H3G34R mutation are sensitive to the combination of vorinostat (a histone deacetylase inhibitor) and PARP-1 inhibitors. The combined treatment increased the phosphorylation of eIF2α (P-eIF2α) relative to each drug alone and enhanced the decrease in cell survival. To explore the role played by increased P-eIF2α in modulating PED-GBM survival and response to treatments, we employed brain-penetrating inhibitors of P-eIF2α dephosphorylation: salubrinal and raphin-1. These drugs increased P-eIF2α, DNA damage, and cell death, similarly affecting the sensitivity of DIPG cells and derived neurospheres to PARP-1 inhibitors. Interestingly, these drugs also decreased the level of eIF2Bϵ (the catalytic subunit of eIF2B) and increased its phosphorylation, thereby enhancing the effect of increased P-eIF2α. Transient transfection with the S51D phosphomimetic eIF2α variant recapitulated the effect of salubrinal and raphin-1 on PED-GBM survival and sensitivity to PARP-1 inhibitors. Importantly, either salubrinal or raphin-1 dramatically increased the sensitivity of DIPG cells to radiation, the main treatment modality of PED-GBM. Finally, PED-GBM was more sensitive than normal human astrocytes to salubrinal, raphin-1, and the treatment combinations described herein. Our results indicate that combinations of histone deacetylase inhibitors and PARP-1 inhibitors should be evaluated for their toxicity and efficacy in PED-GBM patients and point to drugs that increase P-eIF2α or modulate its downstream effectors as a novel means of treating PED-GBM.

Effects of 5-FU on DNA synthesis and cytotoxicity of human lymphocytes induced by IL-2, TGF-beta3 and PGE2.

BACKGROUND: Low 5-fluorouracil (5-FU) concentrations cause a significant increase in DNA synthesis in mitogen-activated human lymphocytes. MATERIALS AND METHODS: We explored 2.5 microM 5-FU-induced DNA synthesis by testing 5-FU activity in hypoxanthine-aminopterin-thymidine (HAT)-containing medium, and its effect on thymidylate synthase (TS) activity and CD25 expression in interleukin (IL)-2-activated human peripheral blood mononuclear cells (PBMCs) and the combined effects with prostaglandin E(2) (PGE(2)) and transforming growth factor (TGF)-beta3. RESULTS: The co-stimulatory effect of 2.5 microM 5-FU on DNA synthesis was abrogated in HAT-cultured medium. 5-FU substantially reduced TS activity by 50% in IL-2-activated PBMCs. 5-FU combined with TGF-beta3 and PGE(2) did not alter their inhibitory effects on IL-2-activated natural killer cell cytotoxicity, but substantially affected increased DNA synthesis of cells cultured in IL-2 and co-cultured with 10 ng/ml TGF-beta3 and 10 microM PGE(2). CONCLUSION: Low 5-FU concentrations increase DNA synthesis in lymphocytes and exert a co-stimulatory activity on TGF-beta3 and PGE(2) modulation of IL-2-activated lymphocytes.

Effects of 5-fluorouracil on human mitogen-activated peripheral blood lymphocytes from healthy individuals.

BACKGROUND: The effect of 5-fluorouracil (5-FU) on activated lymphocytes was explored. MATERIALS AND METHODS: The in vitro effects of 5-FU on DNA synthesis in mitogen-activated lymphocytes from healthy volunteers were compared to those of the antimetabolites doxorubicin, cyclophosphamide and 6-mercaptopurine. These effects were assessed by alterations in the phenotypic profile and the percentage of cells in various phases of the cell cycle, as well as by the secretion of T helper (Th)1 and Th2 cytokines (ELISA). RESULTS: Unlike 5-FU, the other antimetabolites failed to augment DNA synthesis in activated lymphocytes. The effect of 5-FU correlated with an increase in the percentage of cells in the S-phase and caused an increased in CD4+ cells, a decrease in CD56+ cells and a shift of the cytokine secretion pattern from Th2 to Th1. CONCLUSION: 5-FU exhibited a unique effect on DNA synthesis in activated lymphocytes which was accompanied by selective effects on various lymphocyte subpopulations.

In vitro modulation of interleukin-2-mediated human peripheral mononuclear cell proliferation and antitumor cytotoxicity by 5-fluorouracil.

BACKGROUND: Studies on cancer patients undergoing chemotherapy have shown inhibitory effects of anticancer drugs on certain immune activities. MATERIALS AND METHODS: The in vitro effect of 5-(FU) fluorouracil was studied on both lymphocyte proliferation and natural killer (NK) cell antitumor cytotoxicity following incubation of peripheral mononuclear cells (PBMCs) from healthy donors with interleukin (IL)-2, phytohemagglutinin A (PHA) and pokeweed mitogen (PWM). RESULTS: Activation of PBMCs with IL-2, PHA and PWM in the presence of 250 and 2500 microM of 5-FU caused a marked decrease in both the induction of activated natural killer (ANK) cell cytotoxic activity and DNA synthesis, while 2.5 microM increased DNA synthesis by 195%, 58% and 222% for IL-2, PHA and PWM, respectively, more than cells cultured without the drug. No effect of 5-FU was noted on mature ANK cells. CONCLUSION: 5-FU exhibits diverse effects on lymphocyte proliferation and on the generation of ANK antitumor cytotoxic activity.