Overexpression of the p65 Subunit of NF-kB and IkB Mediated Nuclear Sequestration of p53 as Common Events in Philadelphia Positive and Negative Chronic Myeloid Leukemia.

Introduction. Chronic Myeloid Leukemia (CML) is a myeloproliferative disorder caused by the Philadelphia translocation. Rare patients with a clinical presentation of CML are negative for the Ph chromosome. The pathogenesis of Ph negative CML is still unknown. Different reports have demonstrated that the transcription factor NF-kappaB is essential for Bcr-Abl mediated transformation. NF-kappaB is composed of two subunits (mainly p65 and p50) which are retained into the cytoplasm by the inhibitory protein IkappaB-alpha. Different stimuli trigger IkappaB degradation and nuclear translocation of NF-kappaB, where it mediates the transcription of different genes involved in cellular proliferation, transformation and resistance to apoptosis.

Aim of the work. We have evaluated the role of NF-kappaB in primary chronic myeloid leukemia samples both positive and negative for the Philadelphia chromosome.

Methods. Bone marrow samples of 10 chronic myeloproliferative disorders (6 Ph positive and 4 Ph negative CML), 1 CML blast phase, 2 Acute Myeloid Leukemia and 3 healthy donor have been collected at diagnosis. Each sample has been lysed to obtain cytosolic and nuclear extracts. Western blot have been performed to evaluate the expression of p65 and the regulatory protein IkappaB-alpha. DNA binding activity of NF-kappaB has been measured with an ELISA method (TransAM). To inhibit NF-kB, primary cells have been incubated with 1 microM MG-132, 90 microM Resveratrol and 5 microM Bay11-7082 or have been infected with a lentivirus construct containing dsRNA directed against the p65 subunit of NF-kB and a lentivirus expressing a mutated IKB which retains NF-kB inactive in the cytoplasm.

Results. Ph+ and Ph- CML samples express both in the cytosol and in the nucleus higher levels of p65 respect to normal peripheral blood and normal bone marrow samples. In normal samples IkB-alpha is detectable only in the cytosol while in Ph+ and Ph- CML samples it is predominately expressed in the nucleus. DNA binding activity of NF-kappaB is not particularly increased in CML respect to normal bone marrow samples. Only CML blast phase and Acute Myeloid Leukemia show markedly increased DNA binding activity of NF-kappaB. Immunoprecipitates of nuclear and cytosolic IkappaB-alpha have been performed. The pro-apoptotic p53 co-immunoprecipitates with IkappaB-alpha. Treatment with NF-kappaB inhibitors MG-132, Resveratrol and Bay11-7082 disrupts p53-IkBalpha dimer rendering cells susceptible to the apoptotic stimulation induced by Doxorubicine. To confirm these data, primary CML samples have also been infected with a lentivirus construct containing dsRNA directed against p65 and a lentivirus expressing a mutated IKB which retains NF-kB inactive in the cytoplasm. In both cases cells becomes more sensitive to Doxorubicin-induced apoptosis, due to disruption of IkB - p53 dimer.

Conclusion. Nuclear and cytosolic IkappaB-alpha mediated p53 sequestration is a common event in both Ph+ and Ph- CML, which may contribute to the pathogenesis of this disorder due to impairment of p53 pro-apoptotic functions. NF-kB inhibition may represent a powerful strategy to render CML cells more susceptible to apotosis.