Global functional analysis of nucleophosmin in Taxol response, cancer, chromatin regulation, and ribosomal DNA transcription

doi:10.1016/j.yexcr.2006.09.016

Experimental Cell Research

Volume 313, Issue 1, 1 January 2007, Pages 65-76

https://doi.org/10.1016/j.yexcr.2006.09.016 Get rights and content

Abstract

Analysis of lung cancer response to chemotherapeutic agents showed the accumulation of a Taxol-induced protein that reacted with an anti-phospho-MEK1/2 antibody. Mass spectroscopy identified the protein as nucleophosmin/B23 (NPM), a multifunctional protein with diverse roles: ribosome biosynthesis, p53 regulation, nuclear-cytoplasmic shuttling, and centrosome duplication. Our work demonstrates that following cellular exposure to mitosis-arresting agents, NPM is phosphorylated and its chromatographic property is altered, suggesting changes in function during mitosis. To determine the functional relevance of NPM, its expression in tumor cells was reduced by siRNA. Cells with reduced NPM were treated with Taxol followed by microarray profiling accompanied by gene/protein pathway analyses. These studies demonstrate several expected and unexpected consequences of NPM depletion. The predominant downstream effectors of NPM are genes involved in cell proliferation, cancer, and the cell cycle. In congruence with its role in cancer, NPM is over-expressed in primary malignant lung cancer tissues. We also demonstrate a role for NPM in the expression of genes encoding SET (TAF1β) and the histone methylase SET8. Additionally, we show that NPM is required for a previously unobserved G2/M upregulation of TAF1A, which encodes the rDNA transcription factor TAF_I48. These results demonstrate multi-faceted functions of NPM that can affect cancer cells.

Introduction

During our analysis of Taxol-induced protein and gene changes, we observed the G2/M induction of a protein that cross-reacts with an antibody produced against phosphorylated MEK1/2 (pMEK). This protein is smaller than pMEK and migrates as a 37 kDa moiety. After extensive biochemical analysis, this band was identified as nucleophosmin (numatrin, B23). This revelation largely coincided with two published report showing the same [1], [2]. The induction of NPM by Taxol is intriguing, thus this report pursues the functional relevance of NPM in cancer and its global effects on gene profiles.

The relevance of NPM is underscored by the attention it has received from researchers concerned with very different aspects of cell biology. Initial research on the protein centered on its nucleolar distribution and role in ribosome synthesis, though alternate unrelated roles for NPM have also been uncovered [3], [4], [5]. Among these are its capacity to act as a chaperone for nuclear-cytoplasmic shuttling and its participation in the ARF-p53 tumor suppressor pathway [6], [7], [8], [9], [10]. A recent report has also implicated nucleophosmin in the control of transcription by chromatin remodeling [11]. These are exciting aspects of NPM research, but the protein is chiefly implicated in the regulation of cell proliferation and transformation. A more thorough examination of its involvement in these latter two processes is critical to understanding the significance of NPM to the cell.

NPM has long been implicated in cellular transformation, particularly in leukemias and lymphomas. These types of cancer are typically characterized by chromosomal abnormalities such as translocations, which often result in fusion proteins combining the N-terminus of NPM with one of several genes that disrupt its localization and function, including the retinoic acid receptor α (RARα), myelogenous leukemia factor 1 (MLF-1), and the anaplastic lymphoma kinase (ALK) [12]. Recent studies have also identified mutations in the C-terminus of NPM that are associated with acute myelogenous leukemia even in the absence of chromosome rearrangement [13]. Moreover, NPM is abundant in certain hepatomas, and overexpression has been shown to promote the transformation of NIH3T3 cells [14], [15]. The dysregulation of NPM in cancer cells highlights its importance in regulating cell proliferation.

Proliferation depends on ribosome synthesis, and evidence for NPM's role in this process has been accumulating for over two decades [16]. Several lines of evidence have pointed to a role for NPM in the maturation of pre-ribosomal RNA, including its interaction with pre-rRNA particles in cell lines, its in vitro ribonuclease activity, and its localization to the granular region of the nucleolus, where pre-RNA processing takes place. Intriguingly, NPM is also found in the dense fibrillar area of the nucleolus, where transcription of rDNA occurs, suggesting that NPM may play a role not only in the maturation of pre-rRNAs but in their transcription as well [4], [5], [17], [18], [19].

NPM is also important to the segregation of chromosomes and the physical division of daughter cells. During mitosis, the microtubule organizing center duplicates in an NPM-dependent fashion to form the centrosomes, poles from which the mitotic spindle extends [20]. Knockout studies have demonstrated that the absence of NPM results in abnormal accumulation of centrosomes in mouse embryonic fibroblasts [21].

Since we observed Taxol-induced NPM and likely post-translational modification of NPM during mitosis, we focused our attention on its differential functions at that stage of the cell cycle in comparison to interphase. To do so we employed the chemotherapeutic agent Taxol. The primary chemotherapeutic value of Taxol is its ability to bind the β-subunit of tubulin and suppress microtubule dynamics. This prevents dissolution of the mitotic spindle, arresting proliferating cells in mitosis and eventually resulting in their death [22]. While its pro-apoptotic effect is of great clinical importance, Taxol is often used in the laboratory as a mitotic-arresting reagent. Shorter periods of Taxol exposure were used to halt progression of the cell cycle while minimizing cytotoxicity. This allowed us to study the mitosis-specific role of NPM without generating confounding effects such as apoptosis. To understand the role of NPM in transformed cells, we performed our experiments in the H157 cell line, which is derived from advanced non-small cell lung carcinoma (NSCLC).

We reasoned that depletion of nucleophosmin would have consequences on several known and/or novel functions, and that these consequences would be reflected at the level of gene transcription. It is also possible that modification prevents activity of nucleophosmin. We studied NPM function during the cell cycle by depleting it with NPM-using specific small interference siRNA (siRNA) oligonucleotides, then treating cells with Taxol to arrest them in mitosis. After examining the effect of NPM-knockdown on cell cycle progression and apoptosis, we performed large scale microarray analysis to investigate global changes in gene expression. Based on previous studies, we expected NPM to be crucial to cell cycle progression. We found novel roles for NPM in regulating the expression of genes associated with cell proliferation and the progression of cancer. We also show that NPM is upregulated in cancerous tissue, suggesting that it can be used as a marker for NSCLC. Additionally, we present evidence that NPM modulates the transcription of pre-rRNAs by enhancing the transcription of TAF1A, which encodes a component of the RNA Polymerase I machinery. This finding supports the supposition that not only is NPM important the maturation of pre-rRNAs but in their transcription as well.

Section snippets

Cell lines and reagents

The H157 human lung carcinoma line was obtained from the American Type Culture Collection (ATCC), and cultured in RPMI 1640 media (Gibco) with 8% FBS, 10 units/ml penicillin, and 100 μg/ml streptomycin. Cells were maintained at 37°C with 5% CO₂. Paclitaxel (Sigma) and PMA (Sigma) were maintained in a stock solution in dimethyl sulfoxide (DMSO) (Sigma). Anti-pMEK (#9121), anti-total MEK 1/2 (#9122), anti-pNPM (#3541), and anti-pH3 (#9706) antibodies were purchased from Cell Signaling, NPM

Taxol induces a form of nucleophosmin recognized by an antibody to phospho-MEK1/2

When treated with Taxol, the H157 lung cancer cell line arrests in the G2/M phase of the cell cycle. This arrest correlates with increased intensity of a 37 kDa band recognized by anti-phospho-MEK1/2 antibody (Fig. 1A). However, an immunoblot of total MEK1/2 revealed a band that migrated at the expected size of 44 kDa. The intensity of this band does not fluctuate greatly in Taxol-treated cells. This result initially suggested the possibility of a smaller MEK1/2 isoform specific to mitosis [25]

Discussion

Nucleophosmin is a multifunctional protein with roles in a variety of cellular processes, perhaps most importantly in the regulation of cell cycle progression. NPM has long been studied for its participation in ribosome biosynthesis, a necessary step in cell proliferation. Additional evidence points to further roles for NPM in the cell cycle. NPM fusion proteins are common to acute myelogenous leukemia, a disease characterized by chromosome rearrangement, and NPM is also a known regulator of

Acknowledgments

The authors thank Evangeline Reynolds at the Lineberger Comprehensive Cancer Center Tumor Procurement Facility for providing primary lung carcinoma tissues. This work was supported by an NCI Breast Cancer SPORE program grant.

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^☆: This work was supported by NIH Grant CA-58223.

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Research ArticleGlobal functional analysis of nucleophosmin in Taxol response, cancer, chromatin regulation, and ribosomal DNA transcription☆

Abstract

Introduction

Section snippets

Cell lines and reagents

Taxol induces a form of nucleophosmin recognized by an antibody to phospho-MEK1/2

Discussion

Acknowledgments

Biochem. Biophys. Res. Commun.

Biochim. Biophys. Acta

Cell

J. Biol. Chem.

Cell

J. Biol. Chem.

Cancer Lett.

J. Biol. Chem.

Mol. Cell

Cell

J. Mol. Biol.

J. Biol. Chem.

Phosphorylation regulates nucleophosmin targeting to the centrosome during mitosis as detected by cross-reactive phosphorylation-specific MKK1/MKK2 antibodies

Biochem. J.

The ribonuclease activity of nucleolar protein B23

Nucleic Acids Res

Preferential cleavage in pre-ribosomal RNA byprotein B23 endoribonuclease

Nucleic Acids Res.

Nucleolar protein B23 has molecular chaperone activities

Protein Sci.

Nucleophosmin regulates the stability and transcriptional activity of p53

Nat. Cell Biol.

Physical and functional interactions of the Arf tumor suppressor protein with nucleophosmin/B23

Mol Cell. Biol

ARF impedes NPM/B23 shuttling in an Mdm2-sensitive tumor suppressor pathway

Mol. Cell. Biol.

Human histone chaperone nucleophosmin enhances acetylation-dependent chromatin transcription

Mol. Cell. Biol.

Role of nucleophosmin in embryonic development and tumorigenesis

Nature

Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype

N. Engl. J. Med.

Characterization of the cDNA encoding human nucleophosmin and studies of its role in normal and abnormal growth

Biochemistry

Research Article
Global functional analysis of nucleophosmin in Taxol response, cancer, chromatin regulation, and ribosomal DNA transcription☆