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Carcinogenesis, Vol. 23, No. 1, 93-100, January 2002
© 2002 Oxford University Press

CANCER BIOLOGY

Resistance to UV-induced cell-killing in nucleophosmin/B23 over-expressed NIH 3T3 fibroblasts: enhancement of DNA repair and up-regulation of PCNA in association with nucleophosmin/B23 over-expression

Ming H. Wu1,2, Jei H. Chang2 and Benjamin Y.M. Yung2,3

1 Graduate Institute of Pharmacology, National Yang Ming University, Taiwan, Republic of China and
2 Cancer Biochemistry Laboratory, Department of Pharmacology, College of Medicine, Chang Gung University, 259 Wen-Hwa 1st Road, Kwei-San, Tao-Yuan 333, Taiwan, Republic of China


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Nucleophosmin/B23 was rapidly up-regulated after UV irradiation as p53, PCNA and c-Jun. UV induction of nucleophosmin/B23 was evidently increased at 3 h post-irradiation, and reached a maximum at 12 h, and remained high for at least 24 h. Over-expression of nucleophosmin/B23 made cells more resistant to UV-induced cell growth inhibition and death as compared with control vector-transfected cells through three main observations: cell growth/death percentage determination; clonogenic survival assay; and flow cytometric analysis. Moreover, nucleophosmin/B23 over-expressed cells had a greater capacity to repair UV-damaged reporter plasmid, indicating a higher nucleotide excision repair (NER) activity. Furthermore, PCNA, an essential component for DNA repair machinery, was correlated with nucleophosmin/B23 expression. Both protein level and promoter activity of PCNA were higher in nucleophosmin/B23 over-expressed cells than in control vector-transfected cells. On the other hand, treatment of cells with nucleophosmin/B23 antisense oligonucleotides decreased nucleophosmin/B23 and PCNA proteins, and potentiated the UV-induced cell killing. The effect of PCNA up-regulation may be one of the reasons that nucleophosmin/B23 over-expression made cells resistant to UV-induced growth inhibition and cell-killing.

Abbreviations: CAT, chloramphenicol acetyltransferase; ECL, enhanced chemiluminescence reaction; IRF-1, interferon regulatory factor-1; mAb, monoclonal antibody; NER, nucleotide excision repair; PCNA, proliferating cellular nuclear antigen; PVDF, polyvinylidene difluoride; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; TBST, Tris-buffered saline/Tween 20; UV, ultraviolet; YY 1, Yin Yang 1


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
A major adverse effect of ultraviolet C (UVC, 200–290 nm) irradiation on cells is damage to DNA, leading to either cell death or mutation, which is considered to be one of the initial steps of neoplastic transformation (1,2). However, UV irradiation not only leads to damage of cellular components, but also induces specific cellular reactions. A UV-inducible response, known as `SOS response' was first characterized in E.coli, and was shown to be under regulatory control of the recA locus. Among large set of genes involved are those that lead to enhanced DNA repair, mutagenesis, or inhibition of cell division (3). The UV response in mammalian cells is characterized by transcriptional activation or repression of a specific set of genes. One of the earliest and immediate responses to UV is induction of the proto-oncogenes c-fos and c-jun, which regulate various genes harboring AP-1 sites in their promoters (1). The induction of c-Fos and c-Jun is suggested to protect cells from UV-induced apoptosis (4–6). Additionally, p53 is also rapidly induced following UV irradiation, and the increased p53 is part of a protective response which blocks cell cycle progression to allow time for repair of DNA damage before further synthesis and mitosis (7). Proliferating cellular nuclear antigen (PCNA), one of p53 downstream target gene products, is also readily detected simultaneously with p53 expression after UV irradiation (7). PCNA is a necessary component of DNA repair and replication machinery (8). PCNA plays a role in nucleotide excision repair (NER), the pathway for removal of a wide variety of DNA lesions, not only photoproducts induced by UV light but also chemically-induced bulky lesions (8,9).

Nucleophosmin/B23 (37 kd/pI 5.1) also called protein B23 (10), numatrin (11) or NO38 (12), is a nucleolar phosphoprotein. Nucleophosmin/B23 is more abundant in tumor cells than in normal cells (13), and its synthesis is markedly and promptly increased in association with cellular commitment for mitogensis (11,14). It is suggested that nucleophosmin/B23 plays a potential role as a positive regulator of cell proliferation. Nucleophosmin/B23 also appears to be a multifunctional protein. It binds with Rb and synergistically stimulates DNA polymerase {alpha} activity in vitro (15,16). Additionally, nucleophosmin/B23 binds proteins with nuclear localization signals (17), and has been shown to shuttle between nucleolus and cytoplasm (12). Thus, nucleophosmin/B23 appears to carry ribosomal proteins from cytoplasm to nucleolus. Nucleophosmin/B23 inhibits DNA-binding and transcriptional activity of interferon regulatory factor-1 (IRF-1), which is a tumor suppressor (18). Over-expression of nucleophosmin/B23 in NIH 3T3 cells results in malignant transformation, and thus nucleophosmin/B23 seems to be associated with oncogenic activity (19).

Our recent findings have demonstrated that nucleophosmin/B23 is transcriptionally down-regulated during retinoic acid-induced cellular differentiation (20) and sodium butyrate-induced apoptosis (21) of HL-60 leukemia cells. Down-regulation of nucleophosmin/B23 makes HL-60 cells more susceptible to retinoic acid-induced differentiation (20) and sodium butyrate-induced apoptosis (21). These results indicate that nucleophosmin/B23 plays a role in the regulation of nucleolar function for cellular differentiation and apoptosis.

Searching for genes involved in UV-resistance in human cells with mRNA differential display, Higuchi et al. (22) found that expression of nucleophosmin/B23 mRNA is induced after UV irradiation in UV-resistant cells but not in control UV-sensitive cells. Transfection with nucleophosmin/B23 antisense cDNA makes UV-resistant cells become partially sensitive to UV cell-killing. UV-sensitive cells demonstrate lower expression levels of nucleophosmin/B23 compared with those of normal fibroblast cells (23). Additionally, increased phosphorylation and poly(ADP)ribosylation of nucleophosmin/B23 are observed after X-ray treatment of mammalian cells (24). Nucleophosmin/B23 could be a key molecule involved in regulating the susceptibility of cells to stress. The mechanism that nucleophosmin/B23 makes cells resistant to stress becomes an important question to be addressed.

In the present study, attempts were therefore made to establish stable clones of nucleophosmin/B23 overexpressed cells, and determine how nucleophosmin/B23 over-expressed cells respond to UV-induced cell death. Our results showed that nucleophosmin/B23 over-expressed cells, as compared with control vector-transfected cells, were more resistant to UV-induced growth inhibition and cell death. This resistant effect of nucleophosmin/B23 against UV irradiation was associated with enhancement of DNA repair and up-regulation of PCNA.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Reagent and antibodies
All chemicals and anti-FLAG monoclonal antibody (mAb) were purchased from Sigma (St Louis, MO), except where otherwise indicated. Anti-nucleophosmin/B23 mAb was kindly provided by Dr P.K.Chan (Department of Pharmacology, Balyor College of Medicine, Houston, TX). Anti-p53 polyclonal antibody (pAb) and anti-c-Jun pAb were from Santa Cruz (Santa Cruz, CA). Anti-PCNA mAb was from BD Pharmingen (San Diego, CA), and horse radish peroxidase-conjugated goat anti-mouse IgG antibody was from Promega (Madison, WI). Fluorescein-conjugated affinity-purified goat anti-mouse IgG antibody was from Cappel (Turnhout, Belgium).

Cell culture and UV treatments
NIH 3T3 fibroblasts were cultured in Dulbecco's modified Eagle's medium (Life Technologies, Rockville, MD) supplemented with 10% heat-inactivated bovine calf serum (Hyclone, UT), 0.5% antibiotics and 3.7 g/l sodium bicarbonate in a 5% CO2 humidified incubator at 37°C. Numbers of viable and dead cells were determined by trypan blue exclusion and counted with a hemocytometer. For cell immunofluorescence studies, cells were subcultured on glass slides in Petri dish (Nunc, Denmark) 1 day before use. UV treatments (at 254 nm) were performed with a SpectrolinkerTM XL-1000 (Spectronics, New York, NY). Before UV irradiation, the culture medium was removed, and then fresh medium was added to cells. Cells were harvested at indicated times. For mock-treated control cells, the same procedure was followed without irradiation.

Clonogenic survival
Cells were cultured in 6-well plates (Nunc), and appropriate numbers of cells were seeded in triplicate wells for each stable clone to produce at least 30 clones per well. For control vector-transfected cells, 2 x 103 cells per well were seeded, and for nucleophosmin/B23 over-expressed cells, 1 x 103 cells per well were seeded. UV irradiation or mock irradiation of cells was performed 12 h after seeding. Ten days post-irradiation, clones were stained with 0.5% crystal violet (in 70% methanol) for visualization, and the clones whose diameter was >1 mm were counted. The survival percentage was expressed as the relative seeding efficiency of UV-irradiated versus mock-irradiated cultures.

Immunofluorescence
The immunostaining was performed as previously described (10). NIH 3T3 fibroblasts were grown on slides and fixed in 2% formaldehyde (Merck, Darmstadt, Germany) in PBS (8.5 mM Na2HPO4, 1.6 mM NaH2PO4, 0.145 M NaCl, pH 7.2) for 20 min at room temperature. The cells were permeabilized with acetone (Merck) at –20°C for 3 min. After being washed with PBS three times, the fixed cells were incubated with anti-FLAG mAb (diluted 1:260) at 37°C for 1 h. Cells were then washed three times for 15 min each in PBS and incubated with fluorescein-conjugated affinity-purified goat anti-mouse IgG (diluted 1:30 with PBS) at 37°C for 45 min. The cells were washed three times for 15 min with PBS and mounted in 50% (v/v) glycerol in PBS. The results were examined under a fluorescent light microscope (Zeiss, Germany).

Western blotting
Cells were harvested and washed twice in ice-cold PBS, and then lysed in RIPA buffer (1% Triton X-100, 1% SDS, 20 mM Na2HPO4, 100 mM NaCl, 0.2 mM PMSF). Lysates were boiled in SDS sample buffer (62.5 mM Tris (pH 6.8), 5% ß-mercaptoethanol (Merck), 10% glycerol, 2% SDS, 0.001% bromophenol blue), and then fractionated by 10% SDS–PAGE which was carried out according to protocol as previously described (25). Separated proteins in SDS–PAGE were electrotransferred to Hybond-PVDF membrane (Amersham Pharmacia Biotech, Uppsala, Sweden). The PVDF membrane was then soaked in a blocking solution containing 5% (w/v) non-fat milk in TBST (20 mM Tris, pH 7.5, 0.5 M NaCl, 0.1% (v/v) Tween-20) for 1 h at room temperature. To assess nucleophosmin/B23 levels, the soaked PVDF membrane was then incubated with mAb against nucleophosmin/B23 (diluted 1:5000 in 5% (w/v) non-fat milk in TBST) for 4 h at room temperature, and then washed with TBST three times for 15 min each and incubated in horse-radish peroxidase-conjugated goat anti-mouse IgG antibody (diluted 1:5000 in TBST buffer) at room temperature for 1 h. The membrane was washed three times with TBST buffer for 15 min each. Immunobands were detected by the enhanced chemiluminescence reaction (ECL, Amersham Pharmacia Biotech). Anti-p53 pAb (diluted 1:500), anti-c-Jun pAb (diluted 1:500), or anti-PCNA mAb (diluted 1:2500) was used as the primary antibody to detect p53, c-Jun or PCNA, respectively. Equal loading was assessed by protein concentration determinations using Protein Assay kit (Bio-Rad, Richmond, CA) and by Coomassie blue staining of the gel. Quantification of PCNA and nucleophosmin/B23 (B23) in each stable clone. PCNA, nucleophosmin/B23 and ß-actin immuno-band intensities were determined by densitometic scanning. The values of PCNA, nucleophosmin/B23 were normalized with respect to the intensities of ß-actin. Data were analyzed by Image Gauge analysis software (Fujifilm).

Cell cycle and apoptotic cells analysis
2 x 106 cells were fixed in 70% ethanol (in PBS) on ice for 30 min and then resuspended in PBS containing 40 µg/ml propidium iodide, 0.1% Triton X-100 and 0.1 mg/ml RNase (Roche, Mannheim, Germany). After being incubated for 30 min at 37°C, 2 x 104 cells were analyzed on a FACStar cytofluorometer (FACScan, Beckton-Dickinson, San Jose, CA) equipped with an argon-ion laser at 488 nm. Sub-G1 cells were considered as apoptotic cells (26).

Plasmid
To construct clones that encode FLAG-tagged nucleophosmin/B23 protein, we used full-length nucleophosmin/B23 cDNA in plasmid pET-T7 (which was generously given by Dr P.K.Chan) as a template, and following primers to amplify FLAG-nucleophosmin/B23 cDNA by PCR. The N-terminal primer was 5'-ACC ATG GAC TAC AAA GAC GAT GAC GAC AAG CTT ATG GAA GAT TCG ATG GAC-3'. This primer encoded an AUG translation initiation codon followed by the codons for the eight amino acids in the FLAG epitope (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) and six amino acids from nucleophosmin/B23. The C-terminal primer which contained the BamHI site was 5'-CGC CGC GGA TCC TTA AAG AGA CTT CCT CCA CT-3'. Amplified PCR products were then separated and isolated from 1% agarose gel electrophoresis. The 0.9 kilobase FLAG-nucleophosmin/B23 cDNA was then subcloned into the cloning site of the pCR3.1 vector supplied in the Eukaryotic TA cloning kit (Invitrogene, Carlsbad, CA). The orientation of the cDNA in pCR3.1 vector was determined by nucleotide sequencing using the Sequenase kit (Amersham Pharmacia Biotech). The plasmid clone containing the nucleophosmin/B23 cDNA in the sense orientation was driven by Cytomegalovirus (CMV) immediate-early promoter.

The PCNA promoter plasmid, pD6-CAT (nucleotides –693 to +125) was generously given by Dr Y.C.Liu (Department of Life Science, National Tsing-Hua University, Hsin-Chu, Taiwan). The characteristics of pD6-CAT were described previously (27).

Cell transfection and establishment of stable clones
Transfections were performed using LipofectamineTM Reagent (Life Technologies) method. Before transfection, cells (2 x 105 per well) were seeded in 6-well plates overnight. Plasmid DNA (2.5 µg) and LipofectamineTM Reagent (12.5 µg), each diluted in serum-free medium (150 µl). DNA and LipofectamineTM Reagent were then mixed and incubated for 30 min at room temperature to allow DNA-liposome complexes formation. Cells were rinsed twice with PBS, and replaced to serum-free medium (0.7 ml), and then overlaid with DNA-liposome complexes. After 6 h of incubation at 37°C in CO2 incubator, the DNA-containing medium was replaced by fresh medium containing 10% serum.

For establishment of stable clones, pCR3.1-FLAGB23 (sense) or pCR3.1 vector, was transfected into NIH 3T3 fibroblasts as described above. Two days post-transfection, the transfected cells were distributed in 24-well plates at a number of 500 cells/well and 500 µg/ml G418 (Calbiochem, San Diego, CA) was added for selection of stably transfected clones. After selection with G418 for 3 to 5 weeks, individual clones were expanded to mass cultures and subsequently assayed for nucleophosmin/B23-expression. The transfectants were maintained in culture medium supplemented with 250 µg/ml of G418.

Chloramphenicol acetyltransferase (CAT) and luciferase activity assays
CAT assay was performed by single-phase extraction. The cells were lysed in Reporter Lysis Buffer (Promega, Madison, WI) 24 h after transfection. The cellular lysates were heated at 60°C for 10 min to inactivate endogenous deacetylase activity. Total cellular lysates were diluted to 200 µl with 0.1 M Tris (pH 7.8), and added to 200 µl reaction buffer containing 125 mM Tris (pH 8.0), 5 mM chloramphenicol, 0.2 µCi [3H]acetyl-coenzyme A (Amersham Pharmacia Biotech). The reaction mixture was overlayered with 2 ml of a water-immiscible scintillation fluid Ecoscint OTM (National Diagnostic, Atlanta, GE) and then incubated at 37°C for 2 h to measure DNA repair capacity or for 5 h to measure PCNA promoter activity. The CAT activity was quantified in the scintillation counter (Beckman LSD5000, Palo Alto, CA). For luciferase assay, 10 µl of cell lysate was automatically mixed with the reaction buffer (100 µl of 20 mM tricine, 1.07 mM (MgCO3)4 Mg(OH)2, 2.67 mM MgSO4, 0.1 mM EDTA, 33.3 mM DTT, 0.27 mM coenzyme A, 0.47 mM luciferin (Roche, Mannheim, Germany), 0.53 mM ATP at pH 7.8). Luciferase activity was quantified in a luminomer AutoLunmat LB953 (Berthold, Norwalk, CT). Luciferase activity was normalized to the CAT activity of the same extraction without heat treatment.

DNA repair capacity assay
The pCAT Control vector (Promega) is a nonreplicated plasmid, under control of simian virus 40 promoter and enhancer sequences. The plasmid was treated with or without UV (254 nm) at various doses (0, 500 or 1000 J/m2) and then co-transfected with unirradiated pGL3luc (as an internal control) into NIH 3T3 fibroblasts (using LipofectamineTM Reagent as described above). The cells were lysed in Reporter Lysis Buffer 24 h after transfection, and the CAT activity was detected as described above. Relative CAT activity was expressed as the percent of activity in cells transfected with the UV-irradiated over the undamaged plasmid.

Oligonucleotides
The phosphorothioate analogs of deoxyoligonucleotides corresponding to nucleotides –2 to +18 of the nucleophosmin/B23 cDNA were synthesized in the reverse (5'-GCT ACC TTC TAA GCT ACC TG-3') and the antisense (5'-GTC CAT CGA ATC TTC CAT CG-3') orientations (Sigma–Genosys, Woodlands, TX). The synthetic deoxyoligonucleotides composed the 5'-region of the nucleophosmin/B23 cDNA including the translation initiation codon. These oligonucleotides were dissolved and stored in TE buffer (10 mM Tris, pH 7.5, 10 mM EDTA).

Statistical analysis
Data were expressed as means ± standard deviations (SD) throughout the paper. All experiments were performed independently three times. Statistical analyses were performed with one-way ANOVA test. P values <0.05 were considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Nucleophosmin/B23 is up-regulated after UV irradiation
NIH 3T3 fibroblast is often considered as a non-transformed cell line, and has been applied to studies of cellular response to UV irradiation (4,5). Immunoblotting experiments showed that nucleophosmin/B23 increased rapidly after UV irradiation. UV induction of nucleophosmin/B23 was evidently increased at the 3 h time-point, and reached a maximum at 12 h. Thereafter, nucleophosmin/B23 level remained high for at least 24 h (Figure 1Go). In parallel, UV-inducible proteins including c-Jun, p53 and PCNA were also increased in an acute manner after UV irradiation (Figure 1Go).



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  		Fig. 1. Kinetics of nucleophosmin/B23, p53, PCNA and c-Jun induction following UV irradiation. Cells were harvested and lysed at indicated times post-irradiation. Equal amounts of proteins were separated by 10% SDS–PAGE and blotted onto PVDF membrane. Nucleophosmin/B23 (B23), p53, PCNA, c-Jun and ß-actin were detected by Western blot using their specific antibodies and ECL reaction. The same blot was probed with different antibodies after stripping.

 
The characteristics of nucleophosmin/B23 over-expressed cells
Like c-Jun, p53 and PCNA, nucleophosmin/B23 was rapidly up-regulated after UV irradiation (Figure 1Go). To further investigate whether nucleophosmin/B23 played an important role in cellular response to UV irradiation, stable clones of nucleophosmin/B23 over-expressed cells (S6 and S10) and control vector-transfected cells (V) were established as described in Materials and methods. The ectopic expression of FLAG-tagged nucleophosmin/B23 in these stable clones was examined by immunostaining with anti-FLAG antibody. There were 99% of cells expressing FLAG-tagged nucleophosmin/B23 in nucleolus in nucleophosmin/B23 over-expressed cell lines (S6 and S10). No fluorescence was observed in control vector-transfected cells (data not shown). Nucleophosmin/B23 over-expressed cells showed no morphological changes (data not shown) and grew at a rate similar to that of control vector-transfected cells (Figure 2Go). As shown in a previous report (20), nucleophosmin/B23 over-expressed cells exhibited a slightly higher cell density than control vector-transfected cells or the parental cells (NIH 3T3 fibroblasts) (Figure 2Go).



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  		Fig. 2. The growth curve of nucleophosmin/B23 over-expressed, control vector-transfected and NIH 3T3 cells. Nucleophosmin/B23 over-expressed (S6 and S10), control vector-transfected (V) or parental (3T3) cells (5 x 104 cells/well) were seeded onto 6-well plates with DMEM containing 10% bovine calf serum. Cells were harvested at indicated times. Viable and dead cells were determined by trypan blue exclusion method, and cell numbers were obtained by counting with a hemocytometer. Points, means of triplicates ± SD. *P < 0.05, as compared with NIH3T3 cells at comparable times.

 
Nucleophosmin/B23 over-expressed cells are resistant to UV-induced cell growth inhibition and death
Low dose (20 J/m2) of UV irradiation caused cell growth inhibition (Figure 3AGo), and induced a low percentage of cell death (Figure 3BGo). The growth inhibitory effect of low dose UV irradiation in control vector-transfected cells was more obvious than that in nucleophosmin/B23 over-expressed cells (Figure 3AGo). The growth inhibitory effect was only maintained for 12 h in nucleophosmin/B23 over-expressed cells, but persisted for 24 h in control vector-transfected cells (data not shown). Higher dose (30 J/m2) of UV irradiation caused growth inhibition and cell death (Figure 3C,DGo), as determined by trypan blue exclusion. Nucleophosmin/B23 over-expressed cells were more resistant to UV-induced cell death as compared with control vector-transfected cells. Delayed cell death (>20 h) was observed after UVC treatment, as noted by Pourzand and Tyrrell (2). At 48 h after UV irradiation (30 J/m2), <20% of nucleophosmin/B23 over-expressed cells (~18.4% in S6, and 15.4% in S10 clone), while ~32.6% of control vector-transfected were dead (Figure 3DGo). Clonogenic survival assay was employed to assay the long-term survival of cells after UV irradiation (Figure 4Go). The number of colonies was determined following exposure to increasing doses (20–50 J/m2) of UV. The clonogenic survival of nucleophosmin/B23 over-expressed cells was considerably elevated as compared with control vector-transfected cells. The number of colonies in nucleophosmin/B23 over-expressed cells was about two and five times of those in control vector-transfected cells or parental cells after 30 and 50 J/m2 of UV irradiation, respectively. The resistant effect of nucleophosmin/B23 against UV irradiation was more evident with increasing doses of UV irradiation (Figure 4Go).



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  		Fig. 3. Decreased susceptibility to UV-induced cell growth inhibition and death in nucleophosmin/B23 over-expressed cells. (A, C) The relative growth (% of each untreated cell line) of NIH 3T3 cells (3T3), control vector-transfected cells (V), and nucleophosmin/B23 over-expressed cells (S6 and S10) at 48 h after UV irradiation (20 J/m2 or 30 J/m2 at 254 nm). Cell numbers were obtained by counting with a hemocytometer. *P < 0.05, as compared with the relative growth of NIH3T3 cells which were treated with the same dose of UV at 48 h after UV irradiation. (B, D) The percentage of cell death after UV irradiation. Nucleophosmin/B23 over-expressed cells (S6 and S10) and control vector-transfected cells (V) were treated with UV (20 J/m2 or 30 J/m2 at 254 nm). At 48 h after UV irradiation, cells were harvested and counted with a hemocytometer. Viable and dead cells were determined by trypan blue exclusion method. Values, means of triplicates ± SD. *P < 0.05, as compared with the percentage of cell death in NIH3T3 cells (3T3) which were treated with the same dose of UV at 48 h after UV irradiation.

 


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  		Fig. 4. Effect of nucleophosmin/B23 over-expression on clonogenic survival following UV irradiation. Parental (3T3) cells, control vector-transfected cells (V), and nucleophosmin/B23 over-expressed cells (S6 and S10) were cultured in 6-well plates. Colony formation efficiencies were determined 10 days after UV irradiation (20–50 J/m2). The survival percentage was expressed as the relative seeding efficiency of UV-irradiated versus unirradiated cultures. Bars, means of triplicates ± SD. *P < 0.05, as compared with NIH3T3 cells (3T3) which were treated with the same dose of UV irradiation.

 
To provide more evidence regarding the resistance of nucleophosmin/B23 over-expressed cells, DNA content of cells was analyzed by flow cytometry after 30 J/m2 of UV irradiation. There was a sub-G1 DNA peak in all clones at 24 h after UV irradiation, suggesting an apoptotic response to UV (26). The frequency of such apoptotic cells in control vector-transfected cells or parental cells was ~2–3 fold of that in nucleophosmin/B23 over-expressed cells (Figure 5Go). The results indicated that nucleophosmin/B23 over-expressed cells were more resistant to UV-induced apoptosis than control vector-transfected cells or parental cells.



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  		Fig. 5. Flow cytometry studies of cellular DNA contents after UV irradiation. Parental (3T3) cells, control vector-transfected cells (V), and nucleophosmin/B23 over-expressed cells (S6 and S10) were treated with UV (30 J/m2 at 254 nm). At 24 h after UV irradiation, cells were harvested and fixed with ethanol, and stained with propidum iodidine, as described in Materials and methods. The data are presented as number of cells (Y-axis) having a certain PI fluorescence (DNA content; X-axis). The percentage of sub-G1 cells is indicated in each panel.

 
Nucleophosmin/B23 increases cellular DNA repair activity
DNA repair activity is an important determinant for cell survival when cells encounter DNA damage. To elucidate whether the protective effect of nucleophosmin/B23 against UV irradiation was related to DNA repair, DNA repair ability was analyzed by DNA repair capacity assay. In this assay, a reporter plasmid (pCAT) was UV-irradiated to induce DNA lesions prior to transfection. UV-induced damages provides a strong block to transcription, and CAT expression is reduced and only rescued if the damaged plasmid is repaired. If NER activity is enhanced, the damaged plasmid will be repaired more completely and CAT activity will be elevated. DNA repair capacity was measured by CAT activity that was expressed as the percent of activity in cells transfected with UV-irradiated over that in cells transfected with unirradiated plasmid. At 24 h after transfection, a reduction in CAT activity was observed in control vector-transfected cells, indicating that the CAT plasmid was damaged (Figure 6Go). The CAT activity in control vector-transfected cells was reduced to ~55% and 38% by 500 and 1000 J/m2 of UV irradiation, respectively. On the other hand, DNA repair capacity was elevated by nucleophosmin/B23 over-expression: the CAT activity in nucleophosmin/B23 over-expressed cells, slightly reduced to 80–93%, was significantly higher as compared with control vector-transfected cells (Figure 6Go). This result indicated that NER activity was enhanced by over-expression of nucleophosmin/B23.



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  		Fig. 6. DNA repair capacity was enhanced in nucleophosmin/B23 over-expressed cells. Nucleophosmin/B23 over-expressed cells (S6 and S10) or control vector-transfected cells (V) were co-transfected with a CAT reporter plasmid damaged with the indicated doses of UV (500, 1000 J/m2) and an unirradiated plasmid pGL3luc (as an internal control). At 24 h after transfection, CAT activity was measured and normalized to the luciferase activity of the same sample. DNA repair capacity was the relative CAT activity that was expressed as percent of activity in cells transfected with the UV-irradiated over the unirradiated plasmid. Bars, means of triplicates ± SD. *P < 0.05, as compared with the DNA repair capacity in control vector-transfected cells (V), which were transfected with the same dose of UV damaged reporter plasmid.

 
Nucleophosmin/B23 increases PCNA expression
It was noted that the basal level of PCNA in nucleophosmin/B23 over-expressed cells was higher than that in control vector-transfected cells (Figure 7AGo). To compare precisely the levels of PCNA and nucleophosmin/B23 in each clone, the immuno-band intensities of PCNA and nucleophosmin/B23 were quantified and normalized with the intensities of ß-actin. There were 1.2 and 1.65-fold nucleophosmin/B23 protein in S6 and S10 clones respectively as compared with the V clone. In parallel, the protein level of PCNA was increased to 2.2 and 2.95 fold in S6 and S10 clones respectively, as compared with the V clone (Figure 7AGo). It seemed that the levels of PCNA and nucleophosmin/B23 were correlated with each other.




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  		Fig. 7. PCNA was increased in nucleophosmin/B23 over-expressed cells. (A) Cellular lysates of equal amount of proteins (30 µg/lane) from nucleophosmin/B23 over-expressed cells (S6 and S10) and control vector-transfected cells (V) were applied to 10% SDS–PAGE and then transferred onto PVDF membrane. PCNA, nucleophosmin/B23 (B23), and ß-actin were detected by western blot using their specific antibodies and ECL reactions. The same blot was probed with different antibodies after stripping. (B) PCNA promoter activity was increased in nucleophosmin/B23 over-expressed cells. Nucleophosmin/B23 over-expressed cells (S6 and S10) or control vector-transfected cells (V) were cotransfected with pD6-CAT containing PCNA promoter, and pGL3luc as an internal control. At 24 h after transfection, cellular extracts were prepared and assayed for CAT and luciferase activities as described in Materials and methods. The CAT activity obtained from control vector-transfected cells was normalized to one. Bars, means of triplicates ± SD. *P < 0.05, as compared with PCNA promoter activity of control vector-transfected cells (V) cells. (C) Dosage effect of nucleophosmin/B23 on PCNA promoter activity in NIH 3T3 fibroblasts. NIH 3T3 fibroblasts were seeded into 6 cm dishes to reach 50–60% confluency. Cells were transfected with 2 µg of pD6-CAT (PCNA promoter reporter), 0.1 µg of pCR3.1-Luc (as a control for transfection effciency), indicated doses of nucleophosmin/B23 cDNA plasmid, pCR3.1-FLAGB23 (0–1.5 µg), and pGEX. The total amount of plasmid DNA was kept constant (5 µg) by the addition of pGEX. Twenty-four hours after transfection, cells were harvested. The cell extracts containing equal amounts of proteins were analyzed for CAT activity and luciferase activities. CAT units were normalized to luciferase values to control for transfection efficiency. Fold activation was determined by dividing the CAT activity of each sample by the basal CAT activity in the absence of pCR3.1-FLAGB23. Bars, means of triplicates ± SD. *P < 0.05, as compared with the PCNA promoter activity of cells which were not cotransfected with nucleophosmin/B23 cDNA plasmid (dose of nucleophosmin/B23 cDNA plasmid = 0).

 
To study whether nucleophosmin/B23 increased PCNA expression at transcriptional level or not, PCNA promoter activities were assayed in nucleophosmin/B23 over-expressed cells and control vector-transfected cells. A plasmid (pD6-CAT), containing the PCNA promoter (nucleotides –693 to +125) was linked to CAT gene as a reporter to test PCNA promoter activity (28). Following transient transfection, PCNA-CAT expression in nucleophosmin/B23 over-expressed cells was ~1.65-fold (in S6) to 2.55-fold (in S10) of that in control vector-transfected cells (Figure 7BGo). PCNA promoter activity was increased in nucleophosmin/B23 over-expressed cells. To obtain more evidence that nucleophosmin/B23 significantly increased PCNA promoter activity, pD6-CAT was cotransfected into NIH 3T3 fibroblasts with increasing amounts (0.5 to 1.5 µg) of nucleophosmin/B23 expression plasmid (pCR3.1-FLAGB23). Cotransfection with increasing amounts of pCR3.1-FLAGB23 led to increases in transcriptional activation. Transfection with 0.5 or 1.0–1.5 µg of pCR3.1-FLAGB23 showed 2 or 4 to 5-fold activation of PCNA promoter, respectively (Figure 7CGo). The result indicated that nucleophosmin/B23 is involved in the regulation of PCNA promoter in a dose-dependent manner.

Antisense oligonucleotides were also used to examine the role of nucleophosmin/B23 in PCNA expression and UV sensitivity. Western blot analysis showed that nucleophosmin/B23 and PCNA were decreased in nucleophosmin/B23 antisense oligonucleotide-treated cells (Figure 8AGo). The clonogenic survivals of nucleophosmin/B23 antisense oligonucleotide-treated cells were lower as compared with control (+TE buffer) or reverse oligonucleotide-treated cells after UV irradiation (Figure 8BGo).




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  		Fig. 8. The effect of nucleophosmin/B23 antisense oligonucleotides on cellular proteins and clonogenic survival following UV irradiation. (A) NIH 3T3 cells were treated with 20 µM nucleophosmin/B23 antisense oligonucleotides (AS), 20 µM reverse oligonucleotides (R), or an equal volume of TE buffer (TE) for 48 h. Cells were then washed and lysated with RIPA buffer. Then 30 µg of total cellular proteins were separated by 10% SDS–PAGE, blotted onto PVDF. Nucleophosmin/B23 (B23), PCNA, and ß-actin were detected by western blot using their specific antibodies and ECL reaction. The same blot was probed with different antibodies after stripping. (B) NIH 3T3 cells were treated with 20 µM nucleophosmin/B23 antisense oligonucleotides (AS), 20 µM reverse oligonucleotides (R), or an equal volume of TE buffer (TE) for 48 h prior to UV irradiation. After UV irradiation, cells were seeded onto 6-well plates (1 x 103/well). Colony formation efficiencies were determined 10 days after UV irradiation (20–30 J/m2). The survival percentage was expressed as the relative seeding efficiency of UV-irradiated versus unirradiated cultures. Bars, means of triplicates ± SD. *P < 0.05, as compared with control (+TE buffer) cells under same dose of UV irradiation.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Our present study shows nucleophosmin/B23 is rapidly up-regulated after UV irradiation as p53, PCNA or c-Jun. This implicates that nucleophosmin/B23 may play an important role in cellular response to UV irradiation. In support of this, we further find that over-expression of nucleophosmin/B23 makes cells more resistant to UV irradiation through three main observations: cell growth/death percentage determination, clonogenic survival assay and flow cytometric analysis. It has been known that DNA repair ability is one of the important factors for cellular survival after UV irradiation. Indeed, NER is shown to be enhanced in nucleophosmin/B23 over-expressed cells as compared with control vector-transfected cells. The results indicate that it may be the higher DNA repair capacity that contributes to the increased UV-resistance. Furthermore, nucleophosmin/B23 over-expressed cells show no significant changes in cell cycle distribution. It seems that both cell cycle distribution and cell growth rate are not affected by nucleophosmin/B23 over-expression. The resistant effect of nucleophosmin/B23 against UV irradiation may not be mediated by altering cell cycle distribution nor proliferation rate. Furthermore, nucelophosmin/B23 is increased in both parental (Figure 1Go) and nucleophosmin/B23 over-expressed cells (unpublished data) under UV treatment. The total cellular protein amount of nucleophosmin/B23 is higher in over-expressed cells than in parental cells under UV treatment. The nucleophosmin/B23 over-expressed cells, having higher DNA repair ability, are therefore relatively more resistant to UV-induced cell death and growth inhibition as compared with the parental cells. Taken together, nucleophosmin/B23, associated with DNA repair capacity, participates in cellular response to UV irradiation.

UV-induced DNA damage is repaired by NER pathway that is PCNA-dependent (8). PCNA plays a multifunctional role in DNA metabolism. It forms trimer as a ring shape that encircles DNA and slides freely on duplex DNA. In such a way, PCNA can act as a platform for interaction between DNA and many enzymes (or regulatory proteins). PCNA facilitates DNA polymerase {delta} and {varepsilon} to repair UV-induced DNA damage (28). As shown in previous report (29), our present result also shows that PCNA is up-regulated after UV irradiation in a rapid manner. Furthermore, PCNA protein expression and promoter activity are shown to be higher in nucleophosmin/B23 over-expressed cells than in control vector-transfected cells. The PCNA promoter activity is also increased by increasing transient expressions of nucleophosmin/B23 in NIH 3T3 cells. Increase of PCNA by nucleophosmin/B23 may facilitate the cellular process to repair UV-induced DNA damage. It has been reported that PCNA over-expression in chronic lymphocytic leukemia may reflect the intrinsic DNA repair activity of leukemic cells and their resistance to chemotherapy (35). We have also demonstrated that nucleophosmin/B23 antisense oligonucleotides transfection decreases protein expression of PCNA and nucleophosmin/B23, and potentiates UV-induced cell-killing in NIH 3T3 cells (Figure 8Go). Similarly, lower protein expression of nucleo-phosmin/B23 and PCNA, and higher sensitivity to UV-induced cell-killing are observed in HeLa cells that are transfected with a plasmid containing nucleophosmin/B23 cDNA in antisense orientation (36). Judging from the amino acid sequence of nucleophosmin/B23, nucleophosmin/B23 does not have any characteristics as a transcriptional factor. Nucleophosmin/B23 may interact with other transcriptional factor(s), to modulate the transcriptional activity of PCNA gene. There are many proteins that interact with nucleophosmin/B23, including transcriptional factors, such as IRF-1 and Yin Yang 1 (YY1) (19,30). The interaction between nucleophosmin/B23 and transcription factor(s) may be an important regulation in PCNA expression. The mechanism of nucleophosmin/B23 involved in increase of PCNA promoter is under current investigation.

Nucleophosmin/B23 is a protein with multifunctions (12,15–17,19–21,30,31). Nucleophosmin/B23 itself may be with DNA repair activity, because many characteristics of nucleophosmin/B23 are associated with DNA kinetics. For example, nucleophosmin/B23 binds nucleic acids cooperatively with a preference for single-stranded over double-stranded DNA, but without preference for either RNA or single-stranded DNA (31). Single-stranded DNA binding proteins are usually considered to be involved in DNA replication, repair, or recombination (32). Moreover, nucleophosmin/B23 is one of the B-cell-specific DNA recombination complex proteins. It participates in DNA recombination of immunoglobulin heavy chain gene by promoting single-stranded DNA reannealing, and jointing two DNA molecules in D-loop formation. It is notable that the DNA reannealing activity of nucleophosmin/B23 is higher than that of E.coli RecA protein (33). Rec A is associated with SOS response which is one of the DNA repair functions in E.coli (3). On the other hand, nucleophosmin/B23 is similar to p53 in some aspects: p53 protein binds preferentially to the ends of single-stranded DNA, and it promotes both DNA renaturation and DNA strand transfer (34). This evidence may be interpreted as the characteristics of nucleophosmin/B23 involved in DNA repair activity.

In conclusion, when cells undergo UV irradiation, nucleophosmin/B23 is rapidly up-regulated. The increased nucleophosmin/B23 itself or through its affected component (such as PCNA) enhances DNA repair ability and rescues cells from UV-killing. Our result suggests that nucleophosmin/B23, associated with regulation of PCNA and DNA repair, plays an important role in cellular susceptibility to UV-induced growth inhibition and cell death.


   Notes
 
3 To whom correspondence should be addressed Email: byung{at}mail.cgu.edu.tw Back


   Acknowledgments
 
We thank Professor Jonathan T.Ou (Emeritus Professor of Microbiology and an instructor of scientific manuscript writing, Chang Gung University) for carefully proof-reading the manuscript. We also thank Dr W.Y.Kuo for her helpful comments on the manuscript. The work was supported by Chang Gung Memorial Hospital Research Funding Grant CMRP 997, National Science Council (R.O.C) Grant NSC89-2320-B-182-085-M46 and National Research Institute of Heath Council (R.O.C) Grant NHRI-GT-EX89S935L.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received April 30, 2001; revised October 10, 2001; accepted October 23, 2001.


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