Low levels of NPM gene expression in UV-sensitive human cell lines

Cancer Letters 153 (2000) 183±188

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Low levels of NPM gene expression in UV-sensitive human cell lines Junko Hirano a, Xiao-Li Wang b, Kazuko Kita c, Yoshinori Higuchi d, Hiroshi Nakanishi b, Katsuhito Uzawa b, Hidetaka Yokoe b, Hideki Tanzawa b, Akira Yamaura d, Hideo Yamamori a, Nobuyuki Nakajima a, Chikako Nishikiori e, Nobuo Suzuki c,* a

Department of First Surgery, School of Medicine, Chiba University, Inohana 1-8-1, Chuo-ku, Chiba city, Chiba 260-8670, Japan Department of Oral Surgery, School of Medicine, Chiba University, Inohana 1-8-1, Chuo-ku, Chiba city, Chiba 260-8670, Japan c Department of Biochemistry, School of Medicine, Chiba University, Inohana 1-8-1, Chuo-ku, Chiba city, Chiba 260-8670, Japan d Department of Neurosurgery, School of Medicine, Chiba University, Inohana 1-8-1, Chuo-ku, Chiba city, Chiba 260-8670, Japan e Department of Dermatology, Graduate School of Medicine, Kyoto University, 54 Kawaharacho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan b

Received 2 June 1999; received in revised form 21 January 2000; accepted 21 January 2000

Abstract Nucleophosmin (NPM) is a major nuclear matrix protein associated with neoplastic growth in various cell types. We recently suggested that expression of the NPM gene is involved in an increased resistance to UV irradiation in human cells against the cell-killing effects of UV (mainly 254nm wavelength far-ultraviolet ray) [Y. Higuchi, K. Kita, H. Nakanishi, X±L. Wang, S. Sugaya, H. Tanzawa, H. Yamamori, K. Sugita, A. Yamaura, N. Suzuki, Biochem. Biophys. Res. Commun. 248 (1998) 597± 602]. In the present study, expression levels of the NPM gene were examined in human cell lines with a high sensitivity to UV cell-killing. Cockayne syndrome patient-derived cell lines, CSAI and CSBI, and the Xeroderma pigmentosum patient-derived cell line, XP2OS(SV), XP13KY, XP3KA, XP6BE(SV), XP101OS and XP3BR(SV), have been investigated for their NPM mRNA expression with Northern blotting analysis. All of these UV-sensitive cells demonstrated lower expression levels compared with those of normal ®broblast cells, FF, or an UV-resistant cell line, UH r-10; quite a lower level of expression in XP205(SV) cells after UV irradiation in contrast to a distinguishable increase in the expression in UV r- cells. These results con®rmed an intimate correlation between degree of UV sensitivity and expression levels of the NPM gene in human cells. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Nucleophosmin gene; UV sensitivity; Cockayne syndrome; Xeroderma pigmentosum

1. Introduction Nucleophosmin (NPM)/B23/numatrin is a nucleolar phosphoprotein which is more abundant in tumor cells than in normal resting cells [1±6]. NPM has been suggested to play a role on various biological func* Corresponding author. Fax: 181-43-226-2041. E-mail address: [email protected] (N. Suzuki)

tions, such as ribosomal synthesis [7] and regulation of cellular mitogenesis in lymphocytes [4]. It has also been reported to be an index of cell growth and cancer development [8,9]. Recently, we suggested the involvement of NPM in UV resistance in a human cell line [10]. We emp1oyed human cell lines differing in their sensitivity to UV cell-killing, RSb and UV r-10, for a comparative study underlying the difference of cellu-

0304-3835/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(00)00370-0

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lar UV-sensitivity [10]. RSb cells were established from human embryonic ®broblast cells doubly infected with Rous sarcoma virus and Simian virus 40 [11] and were highly sensitive to UV cell-killing [12]. UV r-10 cells were established from RSb cells as a variant with increased resistance to UV cell-killing [12,13]. Amounts of NPM mRNA were more abundant in UV r-10 cells compared with those in RSb cells, and UV r-10 cells transfected with antisense cDNA for NPM mRNA became sensitized to UV cell-killing [10]. Thus, it is interesting to examine whether expression levels of the NPM gene are low in UV-sensitive human cell lines. In this study, we investigated NPM expression levels in immortalized CSAI and CSBI cells derived from Cockayne syndrome (CS) patients, and XP2OS(SV), XP13KY, XP3KA, XP6BE(SV), XP101OS and XP3BR(SV) cells from a Xeroderma pigmentosum (XP) patient. Cockayne syndrome is a very rare autosomal recessive, premature-aging syndrome that includes UV photosensitivity, but few case develop into cancer [14,15]. Xeroderma pigmentosum is also an autosomal recessive condition with UV sensitivity in which skin cancer develops with high frequency [15,16]. Cells isolated from CS and XP patients are highly sensitive to UV irradiation. The present results provide further evidence that the NPM gene is involved in the UV susceptibility of human cells.

cells were a human cell line transformed after infection with Rous sarcoma virus and simian virus 40. UV r-10, an UV-resistant variant cell from RSb, and a ®broblast cell line (FF) were established as described elsewhere [13,23]. Cells were cultured in the medium: Eagle's minimal essential medium (Gibco BRL, Grand Island, NY) containing 10% calf serum (Intergen, NY) and antibiotics (100 mg/ml of streptomycin and 100 units/ ml of penicillin G), at 378C in a humidi®ed atmosphere containing 5% CO2.

2. Materials and methods

2.4. Northern blot analysis

2.1. Cells and culture conditions

Four hours after irradiation, total cellular RNA was isolated from the cells by guanidine isothiocyanatephenol-chloroform extraction using TRIZOL reagent (Gibco BRL, Grand Island, NY), and was freed from contaminating chromosomal DNA by RNase-free DNase (GeneHunter, Brooklyn, MA) as described previously [10]. Ten micrograms of DNase-treated RNA was electrophoresed on a denaturing agarose gel (1% agarose, 6.7% formaldehyde) and blotted onto Hybond-N nylon membranes (Amersham, Buckinghamshire, UK). The 809-bp cDNA for NPM mRNA as cloned previously [10] was used as a probe. After UV crosslinking, the membranes were hybridized to probes labeled with 100 mCi of [a± 32P]dCTP (New England

High UV-sensitive cells, such as CSAI, CSBI, XP2OS(SV), XP13KY, XP3KA, XP101OS and RSb have been described elsewhere [12,17±22]. XP6BE(SV) cells were purchased from Nigms Human Genetic Mutant Cell Repository, the University of Medicine and Dentistry of New Jersey (USA), and XP3BR(SV) cells were supplied by Kenneth Kraemer, NIH (USA). Complementation group of these XP cells are as follows; XP13KY(SV): group A, XP13KY: group A, XP3KA: group C XP6BE(SV): group D, XP101OS: group F, XP3R(SV): group G. XP2OS(SV) XP6BE(SV) and XP3BR(SV) cells were cell lines transformed after infection with simian virus 40. RSb

2.2. UV irradiation Cells were plated in 100-mm dishes and grown to subcon¯uency Then, the cells were irradiated with UV (principally 254-nm wavelength) immediately after the medium was removed, and reincubated in the fresh medium for 4 h. The UV dose was selected as 8 J/m 2 because this was the most ef®cient condition for conferring induction of NPM gene expression [10]. Mock cells were treated in the same manner, but without UV irradiation. 2.3. Colony survival measurements Colony formation assay after UV irradiation was carried out as described in an earlier study [12]. The D0 value was the UV dosage required to reduce the colony-forming ability from any point on the exponential portion of the surviving fraction curve to 37% of that point.

J. Hirano et al. / Cancer Letters 153 (2000) 183±188

Nuclear, MA) using a BcaBEST labeling kit (Takara Biomedicals, Osaka, Japan). Hybridization was carried out as previously described [24]. Autoradiography was performed by exposure of the membranes to Kodak X-Omat ®lm (Kodak, Rochester, NY) for 24±72 h. Membranes were stripped and rehybridized to probes for the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene as a control for RNA loading and transfer. The levels of expressed mRNA were quanti®ed using a Fuji BAS 2000 image analyzer.

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sum patient-derived cells expressed NPM mRNA at a lower level than CSAI and CSBI cells. Three nontransformed cells, XP13KY, XP3KA and XP101OS, expressed the lowest expression levels among XP cells tested. 3.3. The induction of NPM mRNA expression by UV irradiation

In colony survival assay, CSAI, CSBI, XP2OS(SV) as well as RSb cells, were con®rmed to be more sensitive to UV irradiation than FF and UV r-10 cells as shown in Table 1

Levels of NPM gene expression after UV-irradiation were also analyzed by Northern blotting. Levels in CSAI, CSBI, XP2OS(SV), XP6BE(SV), XP3BR(SV) and RSb cells were slightly decreased after UV (8 J/m 2) irradiation, compared with those after mock-irradiation (Fig. 3). A great reduction was observed in XP2OS(SV) cells than in the other UV-sensitive cells. In contrast, FF cells showed an enhanced level of NPM expression, and UV r-10 cells showed a marked increase in NPM mRNA expression after UV irradiation (Fig.3).

3.2. NPM mRNA expression levels in various cell lines

4. Discussion

NPM mRNA expression levels in UV-sensitive cells such as CSAI, CSBI, XP2OS(SV), XP13KY, XP3KA, XP6BE(SV), XP101OS, XP3BR(SV) and RSb were investigated compared with normal FF or UV r-10 cells by Northern blotting analysis. Fig. 1 shows a comparison of Northern blot analysis on NPM expression between CSAI and FF cells. Relative NPM mRNA expression levels to GAPDH mRNA levels were lower in CSAI cells than in FF cells. The mRNA expression level of each cell line tested is summarized in Fig. 2. CSAI, CSBI, XP2OS(SV), XP13KY, XP3KA, XP6BE(SV), XP101OS, XP3BR(SV) and RSb cells showed lower levels of NPM expression compared with FF and UV r-10 cells. The Xeroderma pigmento-

UV radiation activates the expression of a wide variety of genes, which are associated with growth control [25,26]. In our recent study, the NPM gene was found to be expressed differently between UVsensitive and -resistant cells [10]. In this study, NPM gene expression was investigated in cells derived from patients with UV-sensitive diseases, CS and XP. The present results showed lower expression levels

3. Results 3.1. Colony survival

Table 1 Results of UV colony survival in various cell lines Cell

D0 (J/m 2)

CSAI CSBI XP2OS(SV) RSb FF UV r-10

2.6 2.7 0.8 2.4 8.8 9.6

Fig. 1. Northern blot analysis of CSAI and FF cells on NPM mRNA expression.

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Fig. 2. Relative expression levels of NPM mRNA in UV-sensitive and -resistant cells. The NPM mRNA levels were divided by GAPDH mRNA levels. The expression level of UV r-10 cells was designated as 1, and the expression levels of other cells are shown as relative levels to UV r-l0 cells.

of NPM mRNA in UV-sensitive cell lines, including CSM, CSBI and all the XP cells examined, such as XP2OS(SV), XPl3KY, XP3KA, XP6BE(SV), XP101OS and XP3BR(SV), compared with normal FF cells and UV-resistant UV r-l0 cells (Figs. 1 and 2). The lower expression levels of NPM mRNA are irrespective of complementation group and transformation. In addition, NPM mRNA expression levels in almost all UV-sensitive cells were decreased after UV irradiation, contrary to the enhancement of levels in FF and UV r-10 cells (Fig. 3). UV irradiation did not result in a decrease in NPM mRNA expression levels in XP3KA and XP101OS cells (Fig. 3). Interestingly, the two cells constitutively expressed lowest levels of NPM mRNA among the cells examined (Fig. 2). These low levels of may be related to the lack of reduction after UV irradiation. UV r-10 cells expressed high levels of NPM mRNA and showed a greater increase in NPM mRNA expression after UV irradiation than its UV-sensitive parental RSb cells, in accordance with a previous report [10]. Thus, from the present results, low levels of NPM gene expression and without UV induction of the expression were

observed not only in RSb cells, but also in other UV-sensitive human cells. It is noticeable that all of the UV-sensitive cells examined in this study had abnormality of DNA repair and/or its-related mechanisms. RSb cells showed a lower capacity for DNA repair synthesis activity when treated with DNA-damaging agents than UV rl0 cells [12]. XP cells also showed a low repair capacity [16], and CS cells were reported to be de®cient in transcription-coupled repair [14,27,28]. Therefore, further examination is necessary to elucidate whether NPM mRNA expression is related to the DNA repair mechanism. On the other hand, NPM may function in regulation of UV cell-killing through interaction with various protein(s), such as HIV-1 Rev protein [29±31], protein C23 [32] and IRF-1 [9]. Our results require further study to search for protein(s), which interact with NPM and regulate sensitivity to UV cell-killing. Thus, the present results further support the idea that the NPM gene is associated with the UV sensitivity of human cells and indicate the necessity to further study its mechanism.

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Fig. 3. Effects on NPM mRNA expression levels after UV irradiation. The mRNA expression levels were divided by GAPDH mRNA levels, and then shown as the expression of NPM mRNA after UV (8 J/m 2) irradiation relative to those after mock irradiation.

Acknowledgements This study was supported in part by a grant-in-aid from the Smoking Research Foundation, `Ground Research for Space Utilization' promoted by NASDA, the Sumitomo Foundation, the 15th Research-Aid Report in Medical Health Science of Meiji Life Foundation of Health and Welfare, Japan Space Forumre, and research grants from the Ministry of Education, Science and Culture, Japan.

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