Involvement of nucleophosmin/B23 in the response of HeLa cells to UV irradiation

Abstract

A large and pleomorphic nucleolus is one of the important characteristics in diagnosing rapidly growing and dividing cancer cells. 1 The nucleolus is the site of assembly and processing of preribosomal particles. 2 Nucleophosmin/B23 (protein B23, NO38, numatrin), one of the major nucleolar phosphoproteins, is more abundant in tumor and growing cells than in normal resting cells. 3 Nucleophosmin/B23 is localized in the granular region of the nucleolus and is associated with preribosomal particles. The putative function of nucleophosmin/B23 is maturation of ribosomal structures. 4,5 Nucleophosmin/B23 is also a mobile nucleolar protein that shuttles between nucleoli and cytoplasm. 6 Recent studies suggest that nucleophosmin/B23 may recruit protein factors (YY1, p120, Gu, Rev) into nucleoli for rDNA transcription or other functions. 7-10 Our previous studies showed that nucleophosmin/ B23 translocated from nucleoli to the nucleoplasm during the stationary phase of growth 11 or during treatment with certain antitumor drugs, particularly the DNA intercalators. 5,12,13 In light of the many potential roles of nucleophosmin/B23, it is conceivable that nucleophosmin/B23 plays a role in the regulation of cell proliferation.We recently demonstrated that nucleophosmin/B23 was transcriptionally downregulated during retinoic acid (RA)-induced cellular differentiation 14 and sodium butyrate (BuONa)-induced apoptosis 15 of HL-60 leukemia cells. The results indicate that nucleophosmin/B23 plays a role in regulation of the nucleolar function for cellular differentiation and apoptosis. Whether nucleophosmin/B23 is a key molecule in regulating susceptibility of tumor cells to chemotherapeutics or induction of cellular differentiation or apoptosis becomes an important question to be addressed. In a search for genes involved in ultraviolet (UV) resistance in human cells by mRNA differential display, Higuchi et al. 16 have found that expression of nucleophosmin/B23 mRNA is induced after UV irradiation in UV-resistant cells but not in control UV-sensitive cells. The biochemical and molecular events involved in the response of cells to UV irradiation may include a modulation of gene expression. It is an implied hypothesis that expression of these genes relates to the cellular capacity for surviving UV irradiation damage. In the present study, attempts were made to elucidate whether and how nucleophosmin/B23 was involved in cellular susceptibility to UV-induced cell killing. Furthermore, proliferating cell nuclear antigen (PCNA) is required for DNA repair 17 and is induced in cells exposed to UV irradiation. 18 Another goal of our study was to determine the correlation of nucleophosmin/B23 with PCNA and DNA repair capacity in UV sensitivity. Our results show that a decrease in nucleophosmin/B23 by transient transfection of plasmid pCR3-ASB23, a construct that expresses nucleophosmin/B23 mRNA in an antisense orientation, makes the cells more sensitive to UV-induced cell killing. DNA repair activity, as measured by unscheduled DNA synthesis...

Nucleolin and nucleophosmin: nucleolar proteins with multiple functions in DNA repair

“…Phenotypic analyses have revealed increased resistance to UV-induced DNA damage following NCL kd (Yang et al 2009) or NPM1 overexpression (Wu et al 2002a), though measurements of UV radiosensitivity following NPM1 kd/deletion have yielded conflicting results (Vascotto et al 2014;Wu et al 2002b). Separate groups have also used a biochemical assay for repair of a UV-irradiated reporter plasmid to demonstrate decreased NER activity following either overexpression of NCL (Yang et al 2009) or depletion of NPM1 (Wu et al 2002b), suggesting that these proteins may have opposing activities in NER regulation.…”

“…Phenotypic analyses have revealed increased resistance to UV-induced DNA damage following NCL kd (Yang et al 2009) or NPM1 overexpression (Wu et al 2002a), though measurements of UV radiosensitivity following NPM1 kd/deletion have yielded conflicting results (Vascotto et al 2014;Wu et al 2002b). Separate groups have also used a biochemical assay for repair of a UV-irradiated reporter plasmid to demonstrate decreased NER activity following either overexpression of NCL (Yang et al 2009) or depletion of NPM1 (Wu et al 2002b), suggesting that these proteins may have opposing activities in NER regulation. It is important to note, however, that neither of these experimental approaches are able to differentiate between direct activities of NPM1/NCL at sites of NER and global NER deficiencies arising indirectly, such as from NPM1/NCL-dependent transcriptional modulation of the NER machinery (Lin et al 2010;Liu et al 2007;Wu et al 2002a;Wu et al 2002b).…”

“…Separate groups have also used a biochemical assay for repair of a UV-irradiated reporter plasmid to demonstrate decreased NER activity following either overexpression of NCL (Yang et al 2009) or depletion of NPM1 (Wu et al 2002b), suggesting that these proteins may have opposing activities in NER regulation. It is important to note, however, that neither of these experimental approaches are able to differentiate between direct activities of NPM1/NCL at sites of NER and global NER deficiencies arising indirectly, such as from NPM1/NCL-dependent transcriptional modulation of the NER machinery (Lin et al 2010;Liu et al 2007;Wu et al 2002a;Wu et al 2002b). Indeed, Wu et al (2002aWu et al ( , 2002b observed that expression levels of PCNA were closely correlated with those of NPM1 in their system, likely explaining the observed effects on NER efficiency.…”

“…It is important to note, however, that neither of these experimental approaches are able to differentiate between direct activities of NPM1/NCL at sites of NER and global NER deficiencies arising indirectly, such as from NPM1/NCL-dependent transcriptional modulation of the NER machinery (Lin et al 2010;Liu et al 2007;Wu et al 2002a;Wu et al 2002b). Indeed, Wu et al (2002aWu et al ( , 2002b observed that expression levels of PCNA were closely correlated with those of NPM1 in their system, likely explaining the observed effects on NER efficiency. Yang et al (2009) did report a direct interaction between NCL and PCNA that was enhanced following UV irradiation, and found that overexpression of PCNA was able to rescue NCL overexpression-induced NER defects.…”

B23 acts as a nucleolar stress sensor and promotes cell survival through its dynamic interaction with hnRNPU and hnRNPA1

“…B23 possesses a nuclear-localization signal and a nuclearexport signal, which allows it to shuttle between nucleus and cytoplasm (Hingorani et al, 2000;Wang et al, 2005). The shuttling property of B23 contributes to its activity in various cellular processes including the transport of pre-ribosomal particles, the response to stress stimuli such as UV irradiation and hypoxia, as well as cell cycle regulation (Yung et al, 1985;Wu et al, 2002;Brady et al, 2004).…”