Recombinant expression, characterization, and quantification in human cancer cell lines of the Anaplastic Large-Cell Lymphoma-characteristic NPM-ALK fusion protein

Abstract

Systemic anaplastic large cell lymphoma (ALcL) is an aggressive t-cell lymphoma most commonly seen in children and young adults. The majority of pediatric ALCLs are associated with the t(2;5)(p23;q35) translocation which fuses the Anaplastic Lymphoma Kinase (ALK) gene with the nucleophosmin (npM) gene. the npM-ALK fusion protein is a constitutively-active tyrosine kinase, and plays a major role in tumor pathogenesis. In an effort to advance novel diagnostic approaches and the understanding of the function of this fusion protein in cancer cells, we expressed in E. coli, purified and characterized human npM-ALK fusion protein to be used as a standard for estimating expression levels in cultured human ALcL cells, a key tool in ALcL pathobiology research. We estimated that npM-ALK fusion protein is expressed at substantial levels in both Karpas 299 and SU-DHL-1 cells (ca. 4-6 million molecules or 0.5-0.7 pg protein per cell; based on our in-house developed NPM-ALK ELISA; LOD of 40 pM) as compared to the ubiquitous β-actin protein (ca. 64 million molecules or 4.5 pg per lymphocyte). We also compared npM-ALK/ β-actin ratios determined by eLiSA to those independently determined by two-dimensional electrophoresis and showed that the two methods are in good agreement.

Selective Elimination of Senescent Cancer Cells by Galacto-Modified PROTACs

“…It was previously reported that MS99 degrades the nucleophosmin (NPM)−anaplastic lymphoma kinase (ALK) fusion protein. 52 Because Karpas 299 cells overexpress NPM-ALK, 65 normal Karpas 299 (n-Karpas 299) and senescent Karpas 299 (s-Karpas 299) cells were employed to evaluate the degradation ability of Gal-MS99. The results demonstrate that Gal-MS99 does not promote degradation of NPM-ALK in n-Karpas 299 cells even at a concentration of 1 μM (Figure 4B), while in s-Karpas 299 cells, this protein is degraded by both MS99 and Gal-MS99 (Figure 4C).…”

Effect of Diet and Oxidative Stress in the Pathogenesis of Lymphoproliferative Disorders

“…Inactivation of tumor suppressors and activation of proto-oncogenes are secondary to mutations, deletions, or chromosomal translocations [ 128 , 129 , 130 ]. The main translocations implicated in the genesis of lymphomas are t(14;18)(q32;q21), associated with follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) [ 131 , 132 ]; t(8;14)(q24;q32), that is found in Burkitt lymphoma (BL) [ 133 ]; t(2;5)(p23;q35), typical of some T/NK-derived anaplastic large cell lymphomas [ 134 ]; and t(11;14)(q13;q32), described in many cases of mantle cell lymphoma (MCL) [ 135 , 136 ].…”