Investigation of the effects of wip1 phosphatase on chemoteraphy induced cellular response on acute lymphoblastic leukemia cells

PPM1/DWip1 (Protein phosphatase 1D magnesium dependent/wild type p53 induced phosphatase) genotoksik stres üzerine DNA hasarıyla p53’e bağlı olarak indüklenen bir serin/treonin fosfatazdır. Ancak Wip1 fosfataz stres ve doku tipine bağlı olarak E2F, CREB, c-Jun ve NF-κB gibi diğer transkripsiyon faktörleriyle de indüklenebilir. Wip1 fosfataz indüklendiğinde p53, Chk1, Chk2, H2AX, ATM/ATR dahil çok sayıda proteini defosforilasyon yoluyla inaktive ederek stres cevaplarını azaltmakta ve sonuç olarak hücre döngüsü kontrol noktaları, senesens ve apoptoziside engellemektedir. Wip1’in özellikle wtp53’ e sahip insan solid tümörlerinde aşırı ifade edildiği, amplifiye olduğu ve mutasyona uğradığı ve böylece bir onkogen gibi davrandığı bilinmektedir. Wip1’in deregülasyonu solid tümörlerde kötü prognozla da ilişkililendirilmiştir. Ancak hematolojk kanserlerde ve özellikle mt-p53’e sahip olan akut lösemilerde Wip1’in aşırı ifade edildiğinde rolünü ve sonuçlarını gösteren kapsamlı bir çalışma henüz bulunmamaktadır. Bu nedenle bu çalışma da Wip1’i aşırı ifade eden ve mt-p53’ e sahip olan akut T lenfoblastik lösemi hücre dizisi (Jurkat) kullanılarak Wip1’ın kemoterapi indüklü hücresel stres cevapları üzerine rolü araştırılmıştır. Öncelikle Jurkat hücrelerinde Wip1 fosfatazın aşırı ekspresse olduğu qRT-PCR ve Western blot analizleriyle konfirm edilmiştir. Wst-1 testi ile etoposid ve doxorubicinin metabolik aktivite üzerinde etkilerinin zamana ve doza bağlı olduğu tespit edilmiştir. Jurkat hücrelerinin etoposid ve doxorubicinle 24 saatlik muamelesi sonucunda apoptozise direnç gösterirken 72 saatlik muamele sonucunda apoptozis oranının arttığı Annexin V ve Caspase3/7 aktivasyon testleri ile belirlenmiştir. Benzer şekilde Jurkat hücrelerinin etoposid ve doxorubicinle 72 saatlik inkübasyon sonucunda senesens oranının da oldukça düşük olduğu tesbit edilmiştir. Sonrasında Jurkat hücrelerinde etoposid ve doxorubicine cevaben DNA hasarı cevabının önemli elemanlarının ATM, ATR, Chk1, Chk2 ve H2AX dahil fosforilasyon düzeyleri WB analiziyle test edildiğinde özellikle ATM ve ATR’nin fosforilasyon düzeylerinin çok düşük olduğu tesbit edilmiştir. Takiben Jurkat hücrelerinde xvi RNA interferansı yöntemiyle Wip1 hedeflendiğinde ise etoposid ve doxorubicine cevaben DDR elemanlarının hepsinin ATM, ATR, Chk1, Chk2 ve H2AX dahil fosforilasyon düzeylerinin arttığı görülmüştür. Wip1’in Jurkat hücrelerinde RNA interferansı ile hedeflenmesi sürpriz bir şekilde AnnexinV ve Caspase 3/7 testi ile gösterildiği gibi apoptozis oranını azaltırken SA-β-gal testiyle gösteridiği gibi senesens oranını artırmıştır. Ayrıca Jurkat hücrelerinde Wip1’in knock-down edilmesinin hücre döngüsü tutuklanması üzerinde etkili olmadığı görülmüştür.Wild-type p53-induced phosphatase 1 (Wip1/PPM1D), is a serin/threonine phosphatase induced upon genotoxic stress by DNA damage in a p53-dependent manner. However depending on the type of stress or tissue Wip1 phosphatase can be also induced by other transcription factors such as E2F, CREB, c-Jun and NF-κB. Upon induction, Wip1 dampens the stress responses by inactivating multiple proteins via dephosphorylation including p53, Chk1, CHK2, H2AX, ATM/ATR consequently abrogates cell cycle checkpoints and inhibits senescence and apoptosis. Wip1/PPM1D is known to be overexpressed, amplified and mutated in human solid tumors hence displaying typical oncogenic properties. Deregulation of Wip-1 is also associated with poor prognosis in various types of human solid tumors. However, in hematological cancers, especially in acute leukemia containing mt p53, so far no data has shown the role of overexpression of Wip1 phosphatase and it’s consequences. Hence, in this study Wip1 overexpressing acute T lymphoblastic leukemia cell line (Jurkat) containing mt p53 was used to investigate the role of Wip1 phosphatase on chemotherapy induced cellular stress responses. Firstly, overexpression of Wip1 was confirmed with q-RT-PCR and Western blot analyses in Jurkat cells. Metabolik activity of Jurkat cells in response to etoposid and doxorubicin was determined by Wst-1 test in a time and dose dependent manner. When analysed for apoptosis in response to doxorubicin and etoposide treatment by Annexin V and Caspase3/7 tests for 24 h Jurkat cells showed resistance to apoptosis whereas they were responsive after 72 h treatment. Similarly when Jurkat cells were tested for induction of senescence in response to etoposide and doxorubicin treatment the amount of senescencent cells were also little. As next, when Jurkat cells were analysed for phosphorylation status of the key lements of DNA damage response (DDR) ATM, ATR, Chk1 and Chk2 as well as H2AX, in response to etoposide and doxorubicin treatment by WB analysis, the results showed that xviii phosphorylation levels of the DDR elements especially ATM and ATR were significantly low. Accordingly targeting Wip1 levels by RNA interference were increased the phosphorylation status of all DDR elements including ATM, ATR, Chk1, Chk2 and H2AX levels in Jurkat cells. Surprisingly, targeting Wip1 by RNA interference decreased the levels of apopotosis in Jurkat cells whereas it was increased the senescence levels as determined by AnnexinV and Caspase 3/7 and SA-β-gal tests, respectively. In addition knock down of Wip1 was not effective on cell cycle arrest in Jurkat cells

Oncogenic WIP1 phosphatase attenuates the DNA damage response and sensitizes p53 mutant Jurkat cells to apoptosis

Wild-type (wt) p53-induced phosphatase 1 (Wip1), encoded by the protein phosphatase, Mg2+/Mn2+ dependent 1D (PPM1D) gene, is a serine/threonine phosphatase induced upon genotoxic stress in a p53-dependent manner. Wip1/PPM1D is frequently overexpressed, amplified and mutated in human solid tumors harboring wt p53 and is thus currently recognized as an oncogene. Oncogenic Wip1 dampens cellular stress responses, such as cell cycle checkpoints, apoptosis and senescence, and consequently increases resistance to anticancer therapeutics. Targeting Wip1 has emerged as a therapeutic strategy for tumors harboring wt p53. However, little is known about the efficacy of Wip1-targeted therapies in tumors lacking p53. The present study aimed to investigate the potential role of oncogenic Wip1 in p53 mutant (mt) Jurkat cells. In the present study, it was demonstrated that p53 mt Jurkat cells exhibited PPM1D/Wip1 gene amplification and expressed relatively high levels of Wip1, as confirmed by gene copy number and RNA expression analysis. In addition, Jurkat cells underwent G2 cell cycle arrest, apoptotic cell death and senescence in response to etoposide and doxorubicin, although the phosphorylation levels of DNA damage response (DDR) elements, including ataxia-telangiectasia mutated, ataxia-telangiestasia and Rad3-related, checkpoint kinase (Chk)1 and Chk2 were significantly low. Accordingly, the targeting of Wip1 phosphatase by RNA interference increased the phosphorylation of DDR elements, but decreased the rate of apoptosis in response to etoposide or doxorubicin in Jurkat cells. The induction of senescence or cell cycle arrest was not affected by the knockdown of Wip1. The results suggest that increased Wip1 expression enhances the apoptotic sensitivity of Jurkat cells in response to chemotherapeutic agents by attenuating DDR signaling. The present study highlights the possible pro-apoptotic role of Wip1 in a p53 mt T-cell acute lymphoblastic leukemia cell line. The data suggest the careful consideration of future treatment strategies aiming to manipulate or target Wip1 in human cancers lacking p53