Publication: Hematolojik malignitelrede gadd45 epigenetik disregülasyonu
Abstract
1.ÖZET: GADD45 gen ailesi hücrede DNA hasarına cevap oluşturulması ve hücre bölünmesinin durdurulması süreçlerinde etkili olan, nükleer proteinler GADD45α,β ve γ’ yı kodlar. Her üç GADD45 gen ailesi üyesi de MTK1 kinazı aktive ederek p38/ c-jun-NH2-kinaz aktivasyonunu ve apoptozu indüklemektedir. Yapılan çalışmalarda GADD45γ’nın tümör hücrelerinin koloni oluşturmasını ve bölünmesini baskıladığı gösterilmiştir. Ayrıca birçok kanser türünde GADD45γ’nın metile durumda bulunduğu ve metilasyonun ekspresyonu baskıladığı gösterilmiştir. Yapılan çalışmanın amacı hematolojik malignitelerde GADD45γ promotorunun metilasyon durumunun belirlenmesi ve metilasyonun ekspresyona etkisinin saptanmasıdır. Bunun için hemotolojik malignite hücre soylarından ve 117 akut miyeloid lösemi (AML), 94 multipl miyelom (MM) teşhisi konmuş hastadan alınan kan ve kemik iliği örneklerinden DNA izolasyonu yapılmıştır. Bisülfit modifikasyonunun ardından örneklerle metilasyon spesifik polimeraz zincir reaksiyonu (MSP) yapılmıştır. Non-Hodgkin lenfoma hücre serileri L–428 ve L–1236, Burkitt lenfoma hücre serisi Raji hücrelerinde GADD45γ promotoru tamamiyle metillenmiş olduğu, AML hücre serileri GDM–1, HL–60, KG1a ve MM hücre serisi OPM–2 hücrelerinde ise kısmen metillenmiş olduğu görülmüştür. İncelenen AML hasta örneklerinde metilasyon saptanmazken 94 MM hasta örneğinden 8’inde metilasyon saptanmıştır. İstatistiksel hesaplamalar sonucunda metilasyon saptanan hastalarda ortalama kalsiyum düzeyinin metilasyon görülmeyenlere oranla daha yüksek olduğu görülmüştür. Ayrıca GADD45γ’nın metile durumda olduğu hastaların ortalama sağ kalım sürelerinin 13,7 ay, genin metillenmemiş olarak bulunduğu hastalarda ise bu sürenin 57,1 ay olduğu görülmüştür. Metilasyonun genin ekspresyonuna etkisinin belirlenmesi amacıyla hücre soyları DNA metiltransferaz (DNMT) inhibitörü DAC ile 96 saat boyunca inkübe edilmiş ve RT-PCR uygulanmıştır. GADD45γ promotorunun tamamiyle metillenmiş durumda bulunduğu L–1236 hücrelerinde DAC uygulaması sonrası genin ekspresyonunda değişiklik olmazken, Raji, L–428, HL–60, KG1a, OPM–2 hücrelerinde DAC uygulaması sonrası genin ekspresyonunda artış gözlemlenmiştir. Elde edilen bulgular hemotolojik malignitelerde GADD45γ geninin tümör tipine bağlı olarak epigenetik mekanizmalarla sessizleştirilebileceğini göstermiştir. GADD45 gen ailesi, GADD45 gamma, epigenetik, DNA metilasyonu, hematolojik malinite 1.
Epigenetic dysregulation of GADD45 in hematologic malignancies GADD45 gene family encodes the nuclear proteins, GADD45α, β and γ, which response to damage in DNA in a cell and stopping cell proliferation. It has been shown from the research conducted in recent years that GADD45γ suppresses the colony formation and proliferation of tumor cells.The purpose of this study is to determine the methylation state of GADD45γ promoter in hemotologic malignities and the effect of methylation on expression. Therefore, DNA isolation is performed on blood and bone marrow samples of 117 acute myeloid leukemia (AML), and 94 multiple myelom (MM) patients and hematologic malignency cell lines . After the bisulfide modification of DNA, methylation specific polymerase chain reaction (MSP) has been performed on the samples. It has been found that, in Hodgkin lymphoma cell lines L-428 and L-1236 and Burkitt lymphoma cell lines Raji, GADD45γ promoter was fully methylated. However in AML cell lines GDM-1, HL-60, KG1a and MM cell lines OPM-2 , GADD45γ promoter has been found to be partially methylated. No methylation was found in AML patient samples whereas 8 of of 94 MM patient samples show promoter methylation. The survival time of the patients with methylated GADD45γ is 13.7 months, whereas the survival time of the patients with unmethylated GADD45γ is 57.1 months. In order to understand the effect of methylation on the gene expression, the cell lines were treated with DNA methyltransferase inhibitor (DAC) for 96 hours and then real-time polymerase-chain reaction (RT-PCR) was performed. In L-1236 cells where GADD45γ promoter was fully methylated, there has been no change in gene expression after DAC treatment whereas in Raji, L-428, HL-60, KG1a and OPM-2 cells, an increase in gene expression has been observed after DAC treatment,. The findings of this study indicate that in hemotoligic malignancies, GADD45γ gene may be silenced in epigenetic mechanisms according to the type of tumor. Key Words: GADD45 gene family, GADD45 gamma, Epigenetic, DNA methylation, heamatological malignancy
Epigenetic dysregulation of GADD45 in hematologic malignancies GADD45 gene family encodes the nuclear proteins, GADD45α, β and γ, which response to damage in DNA in a cell and stopping cell proliferation. It has been shown from the research conducted in recent years that GADD45γ suppresses the colony formation and proliferation of tumor cells.The purpose of this study is to determine the methylation state of GADD45γ promoter in hemotologic malignities and the effect of methylation on expression. Therefore, DNA isolation is performed on blood and bone marrow samples of 117 acute myeloid leukemia (AML), and 94 multiple myelom (MM) patients and hematologic malignency cell lines . After the bisulfide modification of DNA, methylation specific polymerase chain reaction (MSP) has been performed on the samples. It has been found that, in Hodgkin lymphoma cell lines L-428 and L-1236 and Burkitt lymphoma cell lines Raji, GADD45γ promoter was fully methylated. However in AML cell lines GDM-1, HL-60, KG1a and MM cell lines OPM-2 , GADD45γ promoter has been found to be partially methylated. No methylation was found in AML patient samples whereas 8 of of 94 MM patient samples show promoter methylation. The survival time of the patients with methylated GADD45γ is 13.7 months, whereas the survival time of the patients with unmethylated GADD45γ is 57.1 months. In order to understand the effect of methylation on the gene expression, the cell lines were treated with DNA methyltransferase inhibitor (DAC) for 96 hours and then real-time polymerase-chain reaction (RT-PCR) was performed. In L-1236 cells where GADD45γ promoter was fully methylated, there has been no change in gene expression after DAC treatment whereas in Raji, L-428, HL-60, KG1a and OPM-2 cells, an increase in gene expression has been observed after DAC treatment,. The findings of this study indicate that in hemotoligic malignancies, GADD45γ gene may be silenced in epigenetic mechanisms according to the type of tumor. Key Words: GADD45 gene family, GADD45 gamma, Epigenetic, DNA methylation, heamatological malignancy