The NPM-ALK fusion gene, formed by the t(2;5)(p23;q35) translocation in non-Hodgkin's lymphoma, encodes a 75-kDa hybrid protein that contains the amino-terminal 117 amino acid residues of the nucleolar phosphoprotein nucleophosmin (NPM) joined to the entire cytoplasmic portion of the receptor tyrosine kinase ALK (anaplastic lymphoma kinase). Here, we demonstrate the transforming ability of NPM-ALK and show that oncogenesis by the chimeric protein requires the activation of its kinase function as a result of oligomerization mediated by the NPM segment. Sedimentation gradient experiments revealed that NPM-ALK forms in vivo multimeric complexes of approximately 200 kDa or greater that also contain normal NPM. Cell fractionation studies of the t(2;5) translocation-containing lymphoma cell line SUP-M2 showed NPM-ALK to be localized within both the cytoplasmic and nuclear compartments. Immunostaining performed with both polyclonal and monoclonal anti-ALK antibodies confirmed the dual location of the oncoprotein and also indicated that NPM-ALK is abundant within both the nucleoplasm and the nucleolus. An intact NPM segment is absolutely required for NPM-ALK-mediated oncogenesis, as indicated by our observation that three different NPM-ALK mutant proteins lacking nonoverlapping portions of the NPM segment were each unable to form complexes, lacked kinase activity in vivo, and failed to transform cells. However, NPM could be functionally replaced in the fusion protein with the portion of the unrelated translocated promoter region (TPR) protein that activates the TPR-MET fusion kinase by mediating dimerization through its leucine zipper motif. This engineered TPR-ALK hybrid protein, which transformed cells almost as efficiently as NPM-ALK, was localized solely within the cytoplasm of cells. These data indicate that the nuclear and nucleolar localization of NPM-ALK, which probably occur because of transport via the shuttling activity of NPM, is not required for oncogenesis. Further, the activation of the truncated ALK protein by a completely heterologous oligomerization domain suggests that the functionally important role of the NPM segment of NPM-ALK in transformation is restricted to the formation of kinase-active oligomers and does not involve the alteration of normal NPM functions.The oncogenic activation of protein tyrosine kinases (PTKs) through structural alterations produced by chromosomal rearrangements has been frequently associated with human cancer (52, 57). These alterations often produce chimeric genes encoding novel amino-terminal sequences derived from an unrelated locus fused to the kinase catalytic domain of the PTK. The juxtaposition of these novel sequences with truncated receptor tyrosine kinases (RTKs) is believed to produce conformational changes normally generated by ligand binding, resulting in constitutive activation of the RTK catalytic domain and an unregulated mitogenic signal (12,55). A shared feature of the various amino-terminal fusion partners of the truncated PTKs that have been characteri...

ALK (anaplastic lymphoma kinase) is a tyrosine kinase receptor, expressed as part of the chimeric NPM-ALK protein, in anaplastic large cell lymphomas (ALCLs) exhibiting the t(2;5)(p23;q35) translocation. As a result of this translocation, the NPM (nucleophosmin) gene is fused to the portion of the ALK gene encoding its intracytoplasmic segment. In normal mouse tissues, mRNA encoding the Alk receptor has been found only in neural cells, suggesting involvement of this receptor in the development of the nervous system. The purpose of the present study was to examine the presence of ALK transcripts and protein in normal human tissues and a variety of cell lines and human tumors. Emphasis was placed on neuroblastomas because other tyrosine kinase receptors are expressed in human neuroblastomas. Fifty-six cell lines, including 29 lines of neural origin, and lymphoid and nonlymphoid tissue specimens, including 24 neuroblastomas, were investigated for ALK expression, using reverse transcriptase-polymerase chain reaction, Western blotting, and immunohistochemistry. The results confirmed that mRNA encoding ALK protein was not detectable in any normal or neoplastic hematopoietic tissue tested, except for t(2;5)-positive ALCL. The salient finding was that 13 of the 29 cell lines of neural origin and 22 of 24 neuroblastomas were found to express ALK transcripts and ALK protein. However, no correlation was evident between any known prognostic factors and the level of ALK expression.

Controversies abound concerning hematopoietic stem cell transduction by recombinant adeno-associated virus 2 (AAV) vectors. For human hematopoietic cells, we have shown that this problem is related to the extent of expression of the cellular receptor for AAV. At least a small subset of murine hematopoietic cells, on the other hand, does express both the AAV receptor and the coreceptor, yet is transduced poorly. In the present study, we have found that approximately 85% of AAV genomes were present in the cytoplasmic fraction of primary murine c-Kit(+)Lin- hematopoietic cells. However, when mice were injected intraperitoneally with hydroxyurea before isolation of these cells, the extent to which AAV genomes were detected in the cytoplasmic fraction was reduced to approximately 40%, with a corresponding increase to approximately 60% in the nuclear fraction, indicating that hydroxyurea facilitated nuclear transport of AAV. It was apparent, nonetheless, that a significant fraction of the AAV genomes present in the nuclear fraction from cells obtained from hydroxyurea-treated mice was single stranded. We next tested whether the single-stranded AAV genomes were derived from virions that failed to undergo uncoating in the nucleus. A substantial fraction of the signal in the nuclear fraction of hematopoietic cells obtained from hydroxyurea-treated mice was also resistant to DNase I. That AAV particles were intact and biologically active was determined by successful transduction of 293 cells by virions recovered from murine hematopoietic cells 48 hr postinfection. Although hydroxyurea facilitated nuclear transport of AAV, most of the virions failed to undergo uncoating, thereby leading to only a partial improvement in viral second- strand DNA synthesis and transgene expression. A better understanding of the underlying mechanism of viral uncoating has implications in the optimal use of recombinant AAV vectors in hematopoietic stem cell gene therapy.

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