Abstract
Karyotypic analysis of tumor cells from patients with multiple myeloma (MM), as well as MM cell lines, frequently demonstrates numerous complex chromosomal abnormalities. Moreover, new chromosomal translocations into the switch region of the immunoglobulin heavy chain (IgH) gene (chromosome 14q32), often heralds transformation to more aggressive MM. Since DNA double stranded break repair (DSBR) is important in mediating these processes, these data suggest that abnormalities in DSBR could ultimately lead to genomic instability, clonal evolution and disease progression in MM. Truncated variants of Ku86 protein (i.e. Ku86v) have previously been detected in 86% to 100% of freshly isolated patient MM cells. Since, the Ku70/Ku86 heterodimer functions as the regulatory subunit of the DNA repair enzyme, DNA protein kinase (DNA-PK), we and others have been interested in the altered expression and function of Ku86v proteins in genome maintenance in MM. Although a number of studies have suggested that truncated forms of Ku proteins could be artificially generated by proteolytic degradation in vitro in B cells and the K562 chronic myeloid leukemia cell line, we now show using whole cell Western immunoblotting that the RPMI 8226 and SGH-MM5 human MM cell lines consistently express full-length Ku86 as well as at least 2 forms of Ku86v - a C-terminus truncated 69 kDa variant Ku86 protein (Ku86v-N); and an N-terminus truncated 56 kDa Ku86v (Ku86v-C). Expression of full-length Ku86 and Ku86v proteins was confirmed using electrophoretic mobility shift assays (EMSA) that incorporate a Ku86-specific DNA probe. In contrast, Ku86v proteins were not detected in the non-MM K562 cell line, by neither whole cell Western blotting nor EMSA, as was previously reported. These data confirm that MM cell lines contained bona fide Ku86v proteins that were generated intracellularly. However, the expected shorter mRNA transcripts of Ku86v’s were not detected using Northern blotting, indicating that Ku86v’s could have been generated by enzymatic cleavage, i.e. post-translational modification, rather than by alternative splicing. Since protease digestion of DNA protein kinase (DNA-PK) and Ku proteins is enhanced by proteasome inhibition (i.e. bortezomib treatment) in MM cell lines; these data taken in aggregate further suggest that proteolytic enzymes that are capable of digesting Ku proteins are constitutively activated, and possibly accumulate and/or become further activated under proteasome inhibition in MM cells. In order to characterize the functional role for Ku86v, we demonstrate using EMSA that both full-length Ku86 and Ku86v-N, but not Ku86v-C, are capable of binding DNA. Since the DNA binding motifs of Ku86 are located in the N-terminus, and the functional domains are located in the C-terminus, these data support the notion that whilst Ku86v-N binds DNA, it is in fact incapable of regulating DNA repair. By contrast, although Ku86v-C does not bind DNA, it may be capable of regulating other biological processes. Accordingly, we demonstrate that Ku86v-C binds to CDK4, E2F-4, BAX, Bcl2 and p53; suggesting at least a possible role for Ku86v proteins in regulating the growth and survival of MM cells. In conclusion, this study confirms that MM cells generate at least 3 forms of Ku86 protein, and that the processes of genome maintenance and/or myelomagenesis could be functionally regulated by these abnormal Ku86v proteins.
DNA Damage Response in Multiple Myeloma: The Role of the Tumor Microenvironment
