AMPK Activation in TET2 Downregulated Leukemia Cells Upon Glutamine Limitation

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ISSN 2149-2042 e-ISSN 2149-4606

Volume : 39 Issue : 2 Year : 2024

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AMPK Activation in TET2 Downregulated Leukemia Cells Upon Glutamine Limitation [Medeniyet Med J]

Medeniyet Med J. Ahead of Print: MEDJ-59683 | DOI: 10.4274/MMJ.galenos.2024.59683

AMPK Activation in TET2 Downregulated Leukemia Cells Upon Glutamine Limitation

Ahsen Merve BAYRAK¹, Burcu YUCEL²
¹Istanbul Medeniyet University Institute of Graduate Studies, Department of Molecular Medicine, Istanbul, T�rkiye and Istanbul Medeniyet University Faculty of Medicine, Department of Medical Biology, Istanbul, T�rkiye
²Istanbul Medeniyet University Faculty of Medicine, Department of Medical Biology, Istanbul, T�rkiye, Istanbul Medeniyet University, Science and Advanced Technologies Research Center (BILTAM), Istanbul, T�rkiye and Health Institutes of T�rkiye (TUSEB), Turkish Cancer Institute, Istanbul, T�rkiye

Objective: 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 TET2 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.
Methods: 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.
Results: 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.
Conclusions: 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.

Keywords: Glutamine metabolism, TET2 expression, AML, AMPK, shRNA-mediated gene silencing

TET2 Bask�lanm�� L�semi H�crelerinde Glutamine K�s�tlamas�nda AMPK Aktivasyonu

Ahsen Merve BAYRAK¹, Burcu YUCEL²
¹�stanbul Medeniyet �niversitesi, T�p Fak�ltesi, T�bbi Biyoloji Anabilim Dal�, �stanbul, Turkiye
²�stanbul Medeniyet �niversitesi, Bilim ve �leri Teknolojiler Ara�t�rma Merkezi (B�LTAM), �stanbul, Turkiye

Ama�: Metabolik yeniden programlama, kanser h�crelerinin ay�rt edici bir �zelli�idir. Kanser h�creleri, hayatta kalmak ve �o�almak i�in t�m�r mikro�evresinden glukoz ve glutamin al�m�n� art�r�r. Glutamin de novo sentezlenebilmesine ra�men, bir�ok kanser h�cresi hayatta kalabilmek i�in h�cre d�� glutamine ba��ml�d�r. DNA demetilasyonunda g�rev alan TET2 geni, ayn� zamanda bir t�m�r bask�lay�c� gen olarak bilinir. TET2 geni, akut miyeloid l�semi (AML) dahil olmak �zere �e�itli kanserlerde s�kl�kla mutasyona u�rar. �al��mam�z, TET2 geni bask�lanm�� AML h�cre hatt� HL-60 h�creleri ile glutamin metabolizmas� aras�ndaki ili�kiyi ara�t�rmay� ama�lad�.
Y�ntemler: TET2 ekspresyonu ile glutamin s�n�rlamas� aras�ndaki ili�kiyi de�erlendirmek i�in TET2 geni, shRNA plazmitleri kullan�larak HL-60 h�crelerinde bask�land�. TET2-bask�lanm�� HL-60 h�crelerinin h�cre proliferasyonu, normal ve d��k glutamin ortam�ndaki h�cre say�lar� ile hesapland�. mRNA ekspresyonlar�ndaki de�i�iklikler, say�msal ters transkriptaz-polimeraz zincir reaksiyonu kullan�larak ara�t�r�ld�. GLUL, AMPK-α ve P-AMPKα protein ekspresyonu imm�noblotlama ile de�erlendirildi.
Bulgular: D��k glutamin ortam�nda TET2-bask�lanm�� HL-60 h�crelerinin h�cre say�lar� ve h�cre canl�l�� azald�. TET2-bask�lanm�� HL-60 h�crelerinin h�cre canl�l��n�n kontrol h�crelerinden daha y�ksek oldu�u bulundu. D��k glutaminde TET2-bask�lanm�� h�crelerde GLUL mRNA ekpsresyonunun artt�� bulundu. Ayr�ca, d��k glutamin ortam�nda TET2- bask�lanm�� HL-60 h�crelerinde P-AMPKα protein ekspresyonunun artt�� bulundu.
Sonu�lar: Bulgular�m�z, TET2-bask�lanm�� HL-60 h�crelerinin, glutamin yoksunlu�una kar�� daha diren�li olabilece�ini ve d��k glutamin ortam�n�n, HL-60 h�crelerinin glutamin ba��ml�l�� ile ba�lant�l� olarak, glutamin metabolizmas�nda belirli genlerin ekspresyonunu art�rd��n� g�stermektedir. Ek olarak, d��k glutamin ortam�, TET2-bask�lanm�� HL-60 h�crelerinde P-AMPKα protein seviyesini art�rd�.

Anahtar Kelimeler: Glutamin metabolizmas�, TET2 ekspresyonu, AML, AMPK, shRNA arac�l� gen susturma

Corresponding Author: Burcu YUCEL, T�rkiye

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