Over-Expression of the P65 Subunit of NF-kB and Increased DNA Binding Activity of NF-kB Is a Common Event Both in Philadelphia Positive and Negative Chronic Myeloproliferative Disorders.

The chronic myeloproliferative diseases (CMPD) are clonal disorders characterized by increased proliferation of cells from one or more myeloid lineages. The most common CMPD is Chronic Myeloid Leukemia which is characterized by the Philadelphia t(9;22) chromosomal translocation. The pathogenesis of Philadelphia negative CMPD is poorly understood, although the activation of tyrosine kinases appears to be an essential feature. For example, a constitutively activated PDGF receptor tyrosine kinase (FIP1L1-PDGFRA) is involved in some cases of the hypereosinophilic syndrome (HES), which is a form of CMPD characterized by increased proliferation of eosinophils. Different reports have demonstrated that the transcription factor NF-kB is essential for Bcr-Abl mediated transformation. NF-kB is a transcription factor which is composed of two subunits (generally p65 and p50). NF-kB dimers are retained into the cytoplasm by the inhibitory protein IkB. Different stimuli trigger the Serine phosphorylation of IkB and its proteolitc degradation. Free NF-kB translocates into the nucleus where it mediates the transcription of different genes involved in cellular proliferation, transformation and in apoptosis resistance. The aim of this work is to evaluate whether NF-kB is active both in Ph positive and in Ph negative CMPD. Bone marrow samples of 8 myeloproliferative disorders (3 Philadelphia positive CML, 3 Ph negative CML-like, 1 HES, 1 Idiopathic Myelofibrosis) have been collected at the diagnosis. The t(9;22) positive K562 cell line, derived form a CML blast crysis, has been used as a positive cellular control of the following experiments. Each samples have been lysed to obtain cytosolic and nuclear extracts. Western blot have been performed to evaluate the expression of the p65 subunit of NF-kB, the regulatory protein IkB and the antiapoptotic protein Bcl-2, whose expression may be regulated by NF-kB. Subsequently the DNA binding activity of NF-kB have been measured with an ELISA method. Our data shows that in all samples p65 is over-expressed both in the cytosol and in the nucleus respect to normal peripheral blood and normal bone marrow samples. The antiapoptotic Bcl-2 is also detectable by western blot in all pathological samples. In normal samples IkB is detected only in the cytosol and not in the nucleus while in CMPD samples it is expressed predominately in the nucleus. Basal DNA binding activity of NF-kB is increased in all the nuclear samples but not in normal samples. To assess whether NF-kB is directly involved in the control of cell growth and apoptosis, we have developed a stable K562 cell line expressing the super repressor IkB. The SR-IkB is a mutated for of IkB which can not be degraded causing an cytosolic sequestration of NF-kB. This stable cell line has a marked reduction of cell growth and is more sensible to the apoptotic stimuli. The data described above suggest that NF-kB activation may be a common mechanism of transformation in both Ph positive and negative CMPD and that its inhibition may be a powerful targeted molecular therapy.