PROTEASOME INHIBITION AND NOT NF-ΚB INHIBITION INDUCES APOPTOSIS TO RESISTANT CELLS IN GLUCOCORTICOID-INDUCED APOPTOSIS George I Lambrou 1, Apostolos Zaravinos 2, Maria Adamaki 1, Dimitrios Spandidos 2 and Spiros Vlahopoulos st Department of Pediatrics, University of Athens, Choremeio Research Laboratory, Goudi, Athens. 2 Department of Clinical Virology, University of Crete, School of Medicine, Heraklio, Crete, Greece. INTRODUCTION AIM OF STUDY Glucocorticoids (GC) are among the most significant agents in leukemia treatment. Resistance to glucocorticoids is considered crucial for disease prognosis. We have been working with a GC-resistant cell line system which, as reported previously, it exhibits not only resistance to GCs but also the last have induce anti-apoptotic and mitogenic effects on this cell system (George this sentence does not make sense). We are currently trying to find a target molecule that would sensitize this cell system. So far, we have tested the inhibition of the proteasome and the IKK kinase. To identify target molecules that would sensitize GC-resistant cells, while those molecules are required to participate in to the GR-regulated pathway. MATERIALS AND METHODS The cell system used was the CCRF-CEM cell line. Cells were cultured in RPMI-1640 medium with 15% FBS supplement. Cells were treated with various concentrations of prednisolone, a glucocorticoid, MG132 (Sigma-Aldrich) a potent proteasome inhibitor and Bay11 (Sigma- Aldrich), a known IKK kinase inhibitor. Cells were counted every 24h with a NIHON-KOHDEN CellTaq-a cytometer. Samples were taken daily and apoptosis, as defined by DNA fragmentation and cell cycle distribution were estimated using a FC500 flow cytometer (Beckman Coulter). RESULTS DISCUSSION Elucidating the mechanisms of resistance to GC-induced apoptosis is of crucial importance to the therapy of leukemia. We have identified two potent molecules that would possibly play a role in eradicating resistance mechanisms in childhood leukemia. In particular, it appeared that both compounds exhibit an inhibitory effect on cell proliferation at least at the highest concentrations used, indicating that they both act on the cell cycle machinery. Yet, not both have the same effect on apoptosis. The MG132 compound appears to be more effective than the Bay11 compound. This is a strong indication that sensitization to apoptosis is probably regulated through the proteasome pathway and not through the NFkB pathway. It is however, possible that resistance to GC-induced apoptosis is induced through NFkB activation, yet sensitization does not pass through the same pathway regulatory mechanism. This makes the phenomenon more complicate and interesting at the same time. Further investigations are required to elucidate the mechanisms of resistance to glucocorticoids. The CCRF-CEM cell line it is known to exhibit resistance to GC-induced apoptosis. It is also known that the NF-κB, transcription factor is an immediate target of the GR. Also, the proteasome mechanism is closely connected to the GR pathway. It appeared that the present cell system appeared to be sensitive to concentrations of MG132 starting already from 1uM, which is a concentration that approaches the expected drug levels if administered to an organism. Cellular apoptosis, as defined by DNA fragmentation reached 90%, while this effect was already evident from the very first hours of incubation. On the other hand, inhibiting the IKK kinase, and subsequently the NF-κB pathway, by inhibiting the transcription factor to enter the nucleus did not have the same result as, cells reached an apoptosis level of ~70%. Co-incubation of the inhibitors with the GC did not affect the levels of observed cell death for the MG132 factor nor for the Bay11 inhibitor. Interestingly, both compounds manifested an inhibitory effect on cell proliferation, yet the MG132 compound induces apoptosis while Bay11 not in the same extent. Figure 1. Proliferation assay of the CCRF-CEM cell line under Bay11 and MG132 treatments respectively Figure 2. Apoptosis, as measured by DNA fragmentation, of the CCRF-CEM cell line under Bay11 and MG132 treatments respectively. Figure 4. Apoptosis, as measured by DNA fragmentation, of the CCRF-CEM cell line under MG132 and prednisolone incubation. Figure 3. Comparative apoptosis, as measured by DNA fragmentation and trypan blue, of the CCRF-CEM cell line under MG132 treatments.