Bayrak Ahsen Merve, Yucel Burcu
Istanbul Medeniyet University Institute of Graduate Studies, Department of Molecular Medicine, Istanbul, Türkiye.
Istanbul Medeniyet University Faculty of Medicine, Department of Medical Biology, Istanbul, Türkiye.
Medeni Med J. 2024 Sep 30;39(3):161-168. doi: 10.4274/MMJ.galenos.2024.59683.
Metabolic rewiring is a characteristic of cancer cells. Cancer cells require more nutrients for survival and proliferation. Although glutamine can be produced in cells via a series of enzymatic reactions, a group of cancer cells are dependent on extracellular glutamine for survival. TET2 plays a role in DNA demethylation and is a tumor suppressor gene. The gene is frequently mutated in various cancers, including acute myeloid leukemia (AML). Our study aimed to investigate the association between TET2-knockdown AML cell line HL-60 cells and glutamine metabolism.
To evaluate the association between TET2 expression and glutamine limitation, TET2 was downregulated in HL-60 cells using shRNA plasmids. The proliferation of TET2-knockdown HL-60 cells was calculated in normal and glutamine-deficient medium. GLUL mRNA expression was investigated using quantitative reverse transcription polymerase chain reaction and protein levels were evaluated using immunoblotting.
The numbers and viability of TET2-knockdown HL-60 cells were decreased in low glutamine-containing medium, but the viability of TET2-knockdown HL-60 cells was higher than that of control cells. GLUL mRNA expressions were increased in TET2-knockdown cells in low glutamine. In addition, P-AMPKα protein expression was increased in TET2-knockdown HL-60 cells in low glutamine-containing medium.
Our findings indicate that TET2-knockdown HL-60 cells may be more resistant to glutamine deprivation. In glutamine-deficient medium, the mRNA expression of glutamine synthetase is increased, which could be related to glutamine addiction in cells. In addition, low-glutamyl medium increased the P-AMPKα protein level in TET2-knockdown HL-60 cells.
代谢重编程是癌细胞的一个特征。癌细胞需要更多营养物质来生存和增殖。虽然谷氨酰胺可通过一系列酶促反应在细胞内产生，但一组癌细胞依赖细胞外谷氨酰胺来维持生存。TET2在DNA去甲基化中起作用，是一种肿瘤抑制基因。该基因在包括急性髓系白血病（AML）在内的多种癌症中经常发生突变。我们的研究旨在探讨TET2基因敲低的AML细胞系HL-60细胞与谷氨酰胺代谢之间的关联。
为评估TET2表达与谷氨酰胺限制之间的关联，使用shRNA质粒在HL-60细胞中下调TET2。在正常和谷氨酰胺缺乏的培养基中计算TET2基因敲低的HL-60细胞的增殖情况。使用定量逆转录聚合酶链反应研究GLUL mRNA表达，并使用免疫印迹评估蛋白质水平。
在含低谷氨酰胺的培养基中，TET2基因敲低的HL-60细胞的数量和活力降低，但TET2基因敲低的HL-60细胞的活力高于对照细胞。在低谷氨酰胺条件下，TET2基因敲低的细胞中GLUL mRNA表达增加。此外，在含低谷氨酰胺的培养基中，TET2基因敲低的HL-60细胞中P-AMPKα蛋白表达增加。
我们的研究结果表明，TET2基因敲低的HL-60细胞可能对谷氨酰胺剥夺更具抗性。在谷氨酰胺缺乏的培养基中，谷氨酰胺合成酶的mRNA表达增加，这可能与细胞中的谷氨酰胺成瘾有关。此外，低谷氨酰胺培养基增加了TET2基因敲低的HL-60细胞中P-AMPKα蛋白水平。
