Abstract
Purpose: Bruton's tyrosine kinase (Btk) is known to be critical for B-lymphocyte development, proliferation and differentiation of B-cell lineages, convey signal transduction through B cell receptor (BCR). The present study examined the anti-tumor effects of PRIMA-1 on Btk activity and explored the underlying mechanism, such as apoptosis.
Materials and Methods: Western blot analysis and Quantitative real-time polymerase chain reaction were performed to check the effects of PRIMA-1 on Btk expression level in KBM3 and Namalwa cells. Wild-type Btk and Btk-NLS (nuclear targeted Btk) expression plasmids were transfected in COS-7 cells to establish and characterize the role of Btk in apoptosis using fluorescent microscopy and FACS assay.
Results: İn this study, we observed that exposure of acute myelomonocytic leukemia cells to anti-leukemic drug (PRIMA-1) suppressed Btk expression at mRNA and protein level. Consequently, we also noticed a reduction in the expression of Nrf2 and HO-1 proteins. Remarkably, Btk nuclear localization was increased in response to low PRIMA-1 exposure, while higher concentrations of PRIMA-1 suppressed Btk expression. Furthermore, overexpression of nuclear targeted decreased apoptosis and increased cell viability compared to the wild-type Btk.
Conclusion: Our findings suggest that nuclear Btk reduces the cell apoptosis in response to PRIMA-1 exposure through oxidative response via Nrf2 and HO-1.
Keywords
References
- 1. Smith CI, Islam TC, Mattsson PT, Mohamed AJ, Nore BF, Vihinen M. The Tec family of cytoplasmic tyrosine kinases: mammalian Btk, Bmx, Itk, Tec, Txk and homologs in other species. BioEssays : news and reviews in molecular, cellular and developmental biology. 2001;23:436-46.
- 2. Hussain A, Yu L, Faryal R, Mohammad DK, Mohamed AJ, Smith CI. TEC family kinases in health and disease--loss-of-function of BTK and ITK and the gain-of-function fusions ITK-SYK and BTK-SYK. The FEBS journal. 2011;278:2001-10.
- 3. Conley ME, Dobbs AK, Farmer DM, Kilic S, Paris K, Grigoriadou S et al. Primary B cell immunodeficiencies: comparisons and contrasts. Annu Rev Immunol. 2009;27:199-227.
- 4. Mohamed AJ, Yu L, Backesjo CM, Vargas L, Faryal R, Aints A et al. Bruton's tyrosine kinase (Btk): function, regulation, and transformation with special emphasis on the PH domain. Immunol Rev. 2009;228:58-73.
Abstract
Amaç: Bruton’s tirozin kinaz (Btk) , B lenfositlerinin gelişimi,
proliferasyonu ve B hücre serilerinin farklılaşmasında, B hücre reseptörü (BCR)
aracılı sinyal iletiminde kritik rol oynadığı bilinmektedir. Bu çalışmada
PRIMA-1’in Btk aktivitesi üzerindeki antitumor etkisi ve altta yatan apopitoz
mekanizması araştırılmıştır.
Gereç ve
Yöntem: KBM3 ve Namalwa hücrelerinde Btk ekpresyon
seviyesi üzerine PRIMA 1’in etkisini kontrol etmek için kantitatif real time
PCR ve Western Blot analizi yapılmıştır. FACS ve flouresan mikroskobi
kullanılarak apoptozda Btk ‘nın rolünü belirlemek için, wild tip Btk ve Btk-NLS
( Nükleus hedefli Btk) ekpresyon plazmidleri COS-7 hücrelerine transfekte
edildi.
Bulgular: Bu çalışmada, akut myelomonositik lösemi hücrelerine anti lösemik
ilaç uygulanması sonucunda, Btk ekspresyonun mRNA ve protein seviyesinde
baskılanmasını gözlemledik. Böylece Nrf2 ve HO-1 proteinlerinin
ekpresyonlarında bir azalma olduğunu da farkettik. Dikkat çekici bir şekilde
Btk nükleer lokalizasyonu, düşük dozda PRIMA-1 uygulaması ile artıyorken,
yüksek dozda PRIMA -1 uygulaması ile Btk ekspresyonu baskılanıyor. Buna ek
olarak, wild tip Btk ile kıyaslandığında, nükleer hedefli aşırı
ekspresyonda, apoptozisde azalma ve hücre canlılık oranında artış ile
sonuçlanıyor.
Sonuç:
Bulgularımıza göre, PRIMA-1 uygulaması ile Nrf2 ve
HO-1 oksidatif cevap aracılığı ile nükleer Btk, hücre apoptozunu azalmaktadır.
Keywords
References
- 1. Smith CI, Islam TC, Mattsson PT, Mohamed AJ, Nore BF, Vihinen M. The Tec family of cytoplasmic tyrosine kinases: mammalian Btk, Bmx, Itk, Tec, Txk and homologs in other species. BioEssays : news and reviews in molecular, cellular and developmental biology. 2001;23:436-46.
- 2. Hussain A, Yu L, Faryal R, Mohammad DK, Mohamed AJ, Smith CI. TEC family kinases in health and disease--loss-of-function of BTK and ITK and the gain-of-function fusions ITK-SYK and BTK-SYK. The FEBS journal. 2011;278:2001-10.
- 3. Conley ME, Dobbs AK, Farmer DM, Kilic S, Paris K, Grigoriadou S et al. Primary B cell immunodeficiencies: comparisons and contrasts. Annu Rev Immunol. 2009;27:199-227.
- 4. Mohamed AJ, Yu L, Backesjo CM, Vargas L, Faryal R, Aints A et al. Bruton's tyrosine kinase (Btk): function, regulation, and transformation with special emphasis on the PH domain. Immunol Rev. 2009;228:58-73.
Details
| Subjects | Health Care Administration |
|---|---|
| Journal Section | Research |
| Authors | |
| Publication Date | March 31, 2017 |
| Published in Issue | Year 2017 Volume: 42 Issue: 1 |
