Increased expression of a COOH‐truncated nucleophosmin resulting from alternative splicing is associated with cellular resistance to ionizing radiation in HeLa cells

Abstract

We previously demonstrated that transfecting HeLa cells with the 24 kDa basic fibroblast growth factor-2 (FGF-2) isoform dramatically increased cell survival after irradiation. To investigate genes implicated in this radioresistance acquisition, we compared mRNA expression between radioresistant 24 kDa FGF-2-expressing cells (HeLa 3A) and radiosensitive control HeLa PINA cells using the differential display technique. Of the 32 differentially expressed mRNAs, 1 presented a significant homology with a known gene. This 378 bp fragment presented 100% identity with exon 11 and 12 of human nucleophosmin (NPM) but differed by including a part of intron 9 in its 5ℙ end. The differential expression of this fragment was confirmed using an RNase protection assay. We then cloned the entire corresponding mRNA and showed that it contained all the exons of NPM plus intron 9, suggesting that it was a splicing product of the NPM gene. This variant encoded for a 35-amino acid truncated NPM (NPM2). NPM2 expression was increased in HeLa 3A. To investigate NPM2's role in radioresistance acquisition, we transfected HeLa cells with NPM2 cDNA and analyzed survival after irradiation of the clones obtained. After transfection with NPM2, radiosensitive HeLa cells exhibited a dramatic increase in cell survival after irradiation. Taken together, our results demonstrate that expression of a COOH-truncated NPM form resulting from the alternative splicing of NPM mRNA is able to increase cell survival after irradiation and suggests that it might be involved in cellular response to ionizing radiation.

Altered nucleophosmin transport in acute myeloid leukaemia with mutated NPM1: molecular basis and clinical implications

“…As NLS-mediated nuclear import of NPM1wt greatly predominates over export, 41 most NPM1wt resides within the nucleolus (Figure 2). In contrast, the rare NPM1.2 (or B23.2) truncated isoform localizes to nucleoplasm, 8 as it lacks the C-terminus nucleolar-binding domain and cannot, therefore, bind efficiently to the nucleolus. 24 Knowledge of how the nucleophosmin functional domains regulate its steady state cellular traffic helped elucidate the molecular mechanisms underlying aberrant cytoplasmic expression of nucleophosmin in AML with mutated NPM1 (see below).…”

“…NPM1 (B23.1) and NPM1.2 (B23.2) have different subcellular distribution patterns: 5 NPM1 protein is localized only in the nucleolus 6,7 and NPM1.2 mainly in the nucleoplasm. 8 A third variant (accession number NM_199185) lacks an alternate in-frame exon compared with variant 1, resulting in a shorter protein whose functions and expression pattern are unknown.…”

Nucleophosmin: A versatile molecule associated with hematological malignancies

“…Although the biological significance of the short isoform remains unclear, its expression is increased in radiation-insensitive cell lines and the product is localized in the cytoplasm as well as in the nucleus. (4,6) The structural features of NPM consist of an oligomerization domain, a metalbinding motif, a bipartite nuclear localization signal, two Asp/ Glu-rich domains, phosphorylation sites for CDK2 and a nucleor localization signal. (6,7) NPM1 has been recognized by oncologists as a partner gene for various chromosomal translocations: NPM-anaplastic lymphoma kinase (ALK) in anaplastic large cell lymphoma (ALCL) with t(3;5), NPM-RARA in acute promyelocytic leukemia (APL) with t(5;17), and NPM-MLF1 in acute myeloid leukemia (AML)/ myelodysplastic syndrome (MDS) with t(3;5).…”

“…(3) In 2002, a shorter isoform encoding a 259-amino acid protein that differs at the C-terminus was also isolated. (4) The NPM1 gene spans 25 kb, contains 12 exons and maps to chromosome 5q35. (4,5) Exon 8 is frequently skipped, and exon 10 is used only for the short isoform.…”

“…(4) The NPM1 gene spans 25 kb, contains 12 exons and maps to chromosome 5q35. (4,5) Exon 8 is frequently skipped, and exon 10 is used only for the short isoform. Although the biological significance of the short isoform remains unclear, its expression is increased in radiation-insensitive cell lines and the product is localized in the cytoplasm as well as in the nucleus.…”