Global DNA Methylation analysis of imatinib resistant and sensitive K562 cells

Öz

OBJECTIVE: Chronic myeloid leukemia (CML) is a hematological disease which is known by the presence of Philadelphia chromosome (Ph+). BCR-ABL protein is expressed by Ph+ chromosome, represents constant increased tyrosine kinase activity. Imatinib (IMA) is a tyrosine kinase inhibitor (TKI) which is utilized as a first line treatment in CML. Emergence of IMA resistance at some point of therapy leads to treatment failure. DNA methylation is considered to be the most stable epigenetic change and several studies have shown that epigenetic changes may play a role in drug resistance. We investigated the global methylation profile of IMA-sensitive K562S, IMA-resistant K562R and IMA-resistant and adherent K562R (K562R-adh) cells to determine whether epigenetic reprogramming is involved in the resistance to IMA and the change in phenotype due to this resistance. MATERIAL AND METHODS: In this study, morphologically distinct, IMA-sensitive K562S and 5µM IMA-resistant K562R and K562R-adh in-vitro CML cell models were used to analyze the global DNA methylation profile. After DNA was isolated from the cells, global 5mC DNA methylation profiles were investigated by ELISA using equal amounts of DNA. RESULTS: Compared to K562S, the global methylation of K562R showed an increase in DNA methylation profile, but this increase in methylation was not statistically significant. Whereas, a slight hypermethylation was observed in the DNA of the K562R-adh vs K562S and K562R-adh vs K562R which is statistically significant. We observed slight hypermethylation in IMA-resistant cells lines versus to the IMA-sensitive cell line. CONCLUSION: Our observed differences in 5methyl-Cytosine on CpG islands (5mC) in K562S versus K562R and K562R-adh cell lines suggest that the DNA methylation alteration in resistant cells may partly contributed in phenotype switching.

Anahtar Kelimeler

• chronic myeloid leukemia
• Imatinib resistance
• Global methylation
• EMT

İmatinibe dirençli ve duyarlı K562 hücrelerinin global DNA Metilasyon analizi

Öz

Kronik miyeloid lösemi (KML), Philadelphia kromozomunun (Ph+) varlığı ile bilinen hematolojik bir hastalıktır. BCR-ABL proteini Ph+ kromozomu tarafından ifade edilir ve sürekli olarak artmış tirozin kinaz aktivitesi göstermektedir. İmatinib (IMA), KML'de ilk basamak tedavi olarak kullanılan bir tirozin kinaz inhibitörüdür (TKI). Tedavinin bir noktasında IMA direncinin ortaya çıkması tedavi başarısızlığına yol açar. DNA metilasyonu en stabil epigenetik değişiklik olarak kabul edilir ve epigenetik değişikliklerin ilaç direncinde rol oynayabileceği çeşitli çalışmalarda gösterilmiştir. Bu çalışmada, IMA’ya karşı gelişen dirençte ve bu direnç nedeniyle fenotipte görülen değişimde epigenetik yeniden programlamanın rol alıp almadığını belirlemek amacıyla, IMA'ya duyarlı K562S, IMA'ya dirençli K562R ve IMA'ya dirençli ve yapışan K562R (K562R-adh) hücrelerinin global metilasyon profilini araştırdık. GEREÇ VE YÖNTEM: Bu çalışmada, global DNA metilasyon profilini analiz etmek için morfolojik olarak farklılık gösteren, IMA'ya karşı duyarlı K562S ve 5µM IMA'ya karşı dirençli K562R ve K562R-adh in-vitro KML hücre modelleri kullanılmıştır. Hücrelerden DNA izole edildikten sonra, eşit miktardaki DNA’lar kullanılarak ELISA yöntemi ile global 5mC DNA metilasyon profilleri araştırılmıştır. BULGULAR: K562R’nin global metilasyonu K562S'ye göre kıyaslandığında, DNA metilasyon profilinde artış saptanırken, metilasyondaki bu artış istatistiksel olarak anlamlı bulunmamıştır. Buna karşılık, K562R-adh ile K562S ve K562R-adh ile K562R kıyaslandığında, K562R-adh hücrelerinin global DNA metilasyon profilinde K562S ve K562R hücrelerinine göre istatistiksel olarak anlamlı derecede artış saptanmıştır. SONUÇ: K562S, K562R ve K562R-adh hücre hatlarında CpG adalarındaki 5metil-Sitozin (5mC)'de gözlemlediğimiz farklılıklar, dirençli hücrelerdeki DNA metilasyon değişikliğinin fenotip değişimine kısmen katkıda bulunabileceğini düşündürmektedir.

Anahtar Kelimeler

• Kronik miyeloid lösemi
• Imatinib direnci
• Global metilasyon
• EMT

Kaynakça

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