The question as to whether A3 adenosine receptor (A3AR) agonists, N (6)-(3-iodobenzyl)-adenosine-5'-N- methyluronamide (IB-MECA) and 2-chloro-N (6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (Cl-IB-MECA), could exert cytotoxic effects at high concentrations with or without the involvement of A3AR has been a controversial issue for a long time. The initial findings suggesting that A3AR plays a crucial role in the induction of cell death upon treatment with micromolar concentrations of IB-MECA or Cl-IB-MECA were revised, however, the direct and unequivocal evidence is still missing. Therefore, the sensitivity of Chinese hamster ovary (CHO) cells transfected with human recombinant A3AR (A3-CHO) and their counter partner wild-type CHO cells, which do not express any of adenosine receptors, to micromolar concentrations of IB-MECA and Cl-IB-MECA was studied. We observed that IB-MECA and Cl-IB-MECA exhibited a strong inhibitory effect on cell proliferation due to the blockage of cell cycle progression at G1/S and G2/M transitions in both A3-CHO and CHO cells. Further analysis revealed that IB-MECA and Cl-IB-MECA attenuated the Erk1/2 signalling irrespectively to A3AR expression. In addition, Cl-IB-MECA induced massive cell death mainly with hallmarks of a necrosis in both cell lines. In contrast, IB-MECA affected cell viability only slightly independently of A3AR expression. IB-MECA induced cell death that exhibited apoptotic hallmarks. In general, the sensitivity of A3-CHO cells to micromolar concentrations of IB-MECA and Cl-IB-MECA was somewhat, but not significantly, higher than that observed in the CHO cells. These results strongly suggest that IB-MECA and Cl-IB-MECA exert cytotoxic effects at micromolar concentrations independently of A3AR expression.
- MeSH
- adenosin analogy a deriváty farmakologie MeSH
- agonisté adenosinového receptoru A3 farmakologie MeSH
- CHO buňky MeSH
- Cricetulus MeSH
- cytotoxiny farmakologie MeSH
- kontrolní body buněčného cyklu účinky léků MeSH
- lidé MeSH
- mitogenem aktivovaná proteinkinasa 1 antagonisté a inhibitory genetika metabolismus MeSH
- mitogenem aktivovaná proteinkinasa 3 antagonisté a inhibitory genetika metabolismus MeSH
- proliferace buněk účinky léků MeSH
- protoonkogenní proteiny c-akt genetika metabolismus MeSH
- receptor adenosinový A3 genetika metabolismus MeSH
- regulace genové exprese MeSH
- signální transdukce účinky léků MeSH
- transfekce MeSH
- viabilita buněk účinky léků MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The biology of T-cell acute lymphoblastic leukemia (ALL) is characterized by functional pre-T-cell receptor (TCR) signaling. Non-T-cell activation linker (NTAL) is a nonenzymatic transmembrane adaptor molecule that is involved in the proximal signaling of lymphocytes. In our previous work, we found an association between high NTAL expression in T-cell ALL blasts and a favorable response to initial glucocorticoid treatment. In the present study, we confirm our previous observation in an experimental model. In addition, the molecular mechanism of the contribution of NTAL to malignant T-cell ALL blast signaling and to methylprednisolone-induced cell death is analyzed. In the in vitro experiments, we used the T-cell ALL Jurkat cell line (Jurkat/wt) and derived Jurkat cell line with stable NTAL expression (Jurkat/NTAL(+)). Cell signaling and cell death after methylprednisolone treatment and after TCR stimulation were analyzed using flow cytometry, Western blot, and quantitative polymerase chain reaction. Jurkat/NTAL(+) cells are significantly more sensitive to both methylprednisolone treatment and TCR-induced stimulation. In addition, after TCR stimulation, Jurkat/NTAL(+) cells show a higher level of intracellular extracellular signal-regulated kinase 1/2 (ERK) phosphorylation and increased expression of the CD69 activation marker on the cell surface than the Jurkat/wt cells. The ERK inhibitor U0126 almost completely abrogates TCR-induced cell death and, importantly, reverses the sensitizing effect of the NTAL protein on methylprednisolone-induced cell death. In conclusion, NTAL acts as a tumor suppressor that enhances the proximal signaling of leukemic blasts. The key downstream molecule responsible for the biological effect of TCR signaling is ERK. Higher ERK phosphorylation leads to enhanced cell death after TCR stimulation and increases cell sensitivity to methylprednisolone-induced cell death.
- MeSH
- adaptorové proteiny signální transdukční fyziologie MeSH
- apoptóza účinky léků fyziologie MeSH
- butadieny farmakologie MeSH
- CD antigeny metabolismus MeSH
- chemorezistence účinky léků MeSH
- diferenciační antigeny T-lymfocytů metabolismus MeSH
- fosforylace účinky léků MeSH
- inhibitory proteinkinas farmakologie MeSH
- Jurkat buňky účinky léků enzymologie MeSH
- lektiny typu C metabolismus MeSH
- lidé MeSH
- lymfoblastická leukemie-lymfom z prekurzorových T-buněk metabolismus patologie MeSH
- MAP kinasový signální systém účinky léků MeSH
- methylprednisolon farmakologie MeSH
- mitogenem aktivovaná proteinkinasa 1 antagonisté a inhibitory metabolismus MeSH
- mitogenem aktivovaná proteinkinasa 3 antagonisté a inhibitory metabolismus MeSH
- nádorové proteiny antagonisté a inhibitory fyziologie MeSH
- nitrily farmakologie MeSH
- posttranslační úpravy proteinů účinky léků MeSH
- receptory antigenů T-buněk účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- techniky in vitro MeSH
The relationship between signal pathways MEK1/2-ERK1/2 and ATM-p53 in the response to DNA damage is not well understood. The aim of our study was to investigate the effect of mitoxantrone and two protein kinase inhibitors – caffeine (inhibitor of ATM kinase) and U0126 (inhibitor of MEK1/2 kinase) – on MOLT-4 and Jurkat leukaemic cell lines. In this work we show that the inhibition of MEK1/2 is associated with an increased mortality of cells after mitoxantrone treatment. Inhibition of ATM by caffeine delayed mitoxantrone-induced cell death in MOLT-4 cells. Mitoxantrone itself induced cell-cycle arrest and accumulation of the cells in late S and G2/M phase. Inhibition of ATM, but not of MEK1/2, abrogated mitoxantrone-induced cell-cycle arrest. Inhibition of MEK1/2 did not change mitoxantroneinduced up-regulation of p53 and p21, but inhibition of ATM markedly decreased up-regulation of p53 and p21, and p53 phosphorylation on serine 15 and serine 392. It can be concluded that: 1) mitoxantrone- induced phosphorylation of p53 on serine 15 and serine 392 is ATM dependent and MEK1/2-ERK1/2 independent. 2) ATM inhibition by caffeine prevents G2 cell arrest and in p53-positive cells MOLT-4 delays the onset of mitoxantrone-induced cell death. 3) Inhibition of MEK1/2-ERK1/2 cascade potentiates the cytostatic effect of mitoxantrone regardless of the p53 status.
- MeSH
- antitumorózní látky farmakologie MeSH
- apoptóza MeSH
- buněčný cyklus MeSH
- butadieny farmakologie MeSH
- DNA vazebné proteiny antagonisté a inhibitory genetika MeSH
- G2 fáze účinky léků MeSH
- Jurkat buňky MeSH
- lidé MeSH
- mitogenem aktivovaná proteinkinasa 1 antagonisté a inhibitory MeSH
- mitogenem aktivovaná proteinkinasa 3 antagonisté a inhibitory genetika MeSH
- mitoxantron farmakologie MeSH
- nádorové buněčné linie MeSH
- nádorové supresorové proteiny MeSH
- nitrily farmakologie MeSH
- protein-serin-threoninkinasy antagonisté a inhibitory genetika MeSH
- proteiny buněčného cyklu antagonisté a inhibitory genetika MeSH
- signální transdukce MeSH
- Check Tag
- lidé MeSH