Low‐Dose Actinomycin‐D Induces Redistribution of Wild‐Type and Mutated Nucleophosmin Followed by Cell Death in Leukemic Cells

Abstract

Specific mutations involving C-terminal part of the nucleolar protein nucleophosmin (NPM) are associated with better outcome of acute myeloid leukemia (AML) therapy, possibly due to aberrant cytoplasmic NPM localization facilitating induction of anti-NPM immune response. Actinomycin D (actD) is known to induce nucleolar stress leading to redistribution of many nucleolar proteins, including NPM. We analyzed the distribution of both wild-type and mutated NPM (NPMmut) in human cell lines, before and after low-dose actD treatment, in living cells expressing exogenous fluorescently labeled proteins as well as using immunofluorescence staining of endogenous proteins in fixed cells. The wild-type NPM form is prevalently nucleolar in intact cells and relocalizes mainly to the nucleoplasm following actD addition. The mutated NPM form is found both in the nucleoli and in the cytoplasm of untreated cells. ActD treatment leads to a marked increase in NPMmut amount in the nucleoplasm while a mild decrease is observed in the cytoplasm. Cell death was induced by low-dose actD in all the studied leukemic cell lines with different p53 and NPM status. In cells expressing the tumor suppresor p53 (CML-T1, OCI-AML3), cell cycle arrest in G1/G0 phase was followed by p53-dependent apoptosis while in p53-null HL60 cells, transient G2/M-phase arrest was followed by cell necrosis. We conclude that although actD does not increase NPM concentration in the cytoplasm, it could improve the effect of standard chemotherapy in leukemias through more general mechanisms.

Localization of AML-related nucleophosmin mutant depends on its subtype and is highly affected by its interaction with wild-type NPM

“…Balusu et al [25] demonstrated that AML cells expressing mutated NPM are more sensitive to disruptive effects of the inhibitor NSC348884 on NPM oligomerization, in comparison with AML cells expressing NPMwt. Recently, we revealed that the localization of NPMmutA is not exclusively cytoplasmic and that a substantial fraction of NPMmutA still resides in the nucleoli [26]. Moreover, we and other authors [26,27] have shown that due to heterooligomer formation, subcellular distribution of NPMmutA changes when the cells are co-transfected with NPMwt.…”

“…Recently, we revealed that the localization of NPMmutA is not exclusively cytoplasmic and that a substantial fraction of NPMmutA still resides in the nucleoli [26]. Moreover, we and other authors [26,27] have shown that due to heterooligomer formation, subcellular distribution of NPMmutA changes when the cells are co-transfected with NPMwt. In the present work, we used HEK-293T cell system allowing high amplification of transfected plasmids to investigate the localization of various mutation types.…”

“…As described in detail previously [26], gene for nucleophosmin was amplified from cDNA library (Jurkat cells, Origene) by PCR and inserted to vectors peGFP-C2 and pmRFP1-C2 (originally Clontech) designed for expression of protein chimeras with a fluorescent protein connected to the N-terminus of the target protein by standard methods of molecular cloning. NPM mutants were constructed by PCR using extended primers containing mutated part of exon 12 of the NPM1 gene and restriction sites (Table 1).…”

The nucleolus, an ally, and an enemy of cancer cells

“…Furthermore, it prevents neither Act D-induced nucleolar stress nor activation of the p53-dependent and independent pathways leading cells to death by apoptosis or necrosis, respectively. Since NPM status is not so important in the case of leukemias, AML patients with NPMmut may be expected to have a better outcome of therapy, which is associated with cellular localization of NPMmut (Brodská et al 2016). It is worth noting that Act D in combination with inhibitors of histone deacetylase significantly enhances therapeutic effects (Brodská et al 2016), whereas combined with other standard chemotherapeutic agents little contributes to therapeutic efficiency of these agents (Parsons-Doherty et al 2014).…”

“…Since NPM status is not so important in the case of leukemias, AML patients with NPMmut may be expected to have a better outcome of therapy, which is associated with cellular localization of NPMmut (Brodská et al 2016). It is worth noting that Act D in combination with inhibitors of histone deacetylase significantly enhances therapeutic effects (Brodská et al 2016), whereas combined with other standard chemotherapeutic agents little contributes to therapeutic efficiency of these agents (Parsons-Doherty et al 2014). However, although Act D is often successful in treatment of a variety of cancers and it is relatively well tolerated, treatment with Act D is restricted mainly by its toxicity at higher doses, but its application at lower doses in combination with other drugs to synergize therapeutic efficacy allows to minimize its genotoxicity toward normal tissues (Choong et al 2009).…”