View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Elsevier - Publisher Connector

FEBS Letters 580 (2006) 731–735

Expression of APOBEC2 is transcriptionally regulated by NF-jB in human hepatocytes

Tomonori Matsumoto, Hiroyuki Marusawa, Yoko Endo, Yoshihide Ueda, Yuko Matsumoto, Tsutomu Chiba* Department of Gastroenterology and Hepatology, Graduate School of Medicine, Kyoto University, 54 Kawara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan

Received 16 November 2005; revised 21 December 2005; accepted 27 December 2005

Available online 3 January 2006

Edited by Masayuki Miyasaka

mutation that diversifies the variable regions of immunoglob- Abstract Apolipoprotein B mRNA-editing catalytic subunit 2 (APOBEC2) is a member of the nucleic-acid-editing ulin [12,13]. In contrast to these data, little is known . However, the physiological function of APOBEC2 re- about either the specific substrates or the physiological func- mains unclear. We demonstrate that APOBEC2 expression is tions of APOBEC2 [14]. strongly enhanced in response to both tumour necrosis factor-a Previous reports have shown that, like other APOBEC fam- (TNF-a) and interleukin-1b. Inhibition of NF-jB activation ily enzymes, human APOBEC2 has a deaminase activity on invariably blocks TNF-a-induced APOBEC2 expression. The cytidine and deoxycytidine in vitro [14,15]. A critically impor- promoter region of APOBEC2 contains functional NF-jB re- tant function of APOBEC2 might be expected because APO- 0 sponse elements in the 5 untranslated region of the at BEC2 is the most ancient DNA/RNA-editing enzyme 625/616. These results show that APOBEC2 expression is known. The enzyme is highly conserved throughout the verte- regulated by pro-inflammatory cytokines via NF-jB activation brate lineage, whereas the APOBEC1 and APOBEC3 sub- and suggest a possible role of APOBEC2 in the pathophysiology of hepatic inflammation. groups are restricted to mammals [14,16,17]. Although the 2005 Federation of European Biochemical Societies. Published expression of APOBEC2 was originally thought to be re- by Elsevier B.V. All rights reserved. stricted and specifically upregulated in the heart and skeletal muscle, a more recent analysis using a cDNA panel derived Keywords: APOBEC2; TNF-a; NF-jB; Hepatocyte; from multiple tissues has suggested that APOBEC2 (also Pro-inflammatory cytokine termed ARCD1) transcripts are ubiquitous in both human and mouse tissues, including the liver [14,15]. To approach the question of the normal and possibly pathological roles of liver APOBEC2 , we investigated the expression and regulation of human APOBEC2 in human hepatocytes. 1. Introduction

Nucleic acid editing modifies genes and their transcripts in 2. Materials and methods many organisms, contributing to an expansion of the number of gene products without the generation of new genes [1–3]. 2.1. Cell culture and transfection Recently, the apolipoprotein B mRNA-editing enzyme cata- The human hepatoma cell lines HepG2, Hep3B, and Huh7 were cul- lytic subunit (APOBEC) family members have been shown tured in Dulbecco’s modified Eagle’s medium (Gibco-BRL, Tokyo, Ja- to be pivotal in the editing of various genes in humans. The pan) [18]. For plasmid transfections, we used the TransIT transfection APOBEC family contains a group of cytidine deaminases that reagent (Mirus Bio Corporation, Madison, WI). The nuclear factor-jB (NF-jB) inhibitors SN50 and MG132 were purchased from BIOMOL include APOBEC-1, -2, -3A, -3B, -3C, -3D, -3F, and -3G, and International LP (Plymouth Meeting, PA). Tumour necrosis factor-a an activation-induced (AID) [1]. The best- (TNF-a) was from PeproTech EC Ltd. (London, UK), and interleu- characterized example of editing performed by an APOBEC kin-1b (IL-1b) from R&D Systems Inc. (Minneapolis, MN). family member is the editing by APOBEC1 of the mRNA Normal liver tissue was obtained from the non-cancerous tissues of patients with metastatic liver cancer at the time of surgery. Primary hu- encoding apolipoprotein B [4–7]. APOBEC3G plays critical man hepatocytes were isolated and cultured according to a previously roles in the cellular anti-retroviral defence mechanism [8–11]. described protocol [19]. Briefly, the liver specimens were infused The putative DNA-editing enzyme AID, expressed in activated through vascular cut ends with warmed (37 C) Hanks’ solution con- B cells in germinal centres, is essential for the somatic hyper- taining 0.5 mM EDTA (Sigma, St. Louis, MO) and 1 M HEPES (Wako Pure Chemical Industries, Osaka, Japan) using a syringe. The tissue samples were then subjected to enzyme digestion with 0.05% col- *Corresponding author. Fax: +81 75 751 4303. lagenase (Wako). The cell suspension was passed through double lay- E-mail address: [email protected] (T. Chiba). ers of sterile cotton gauze and filtered through a single 75 lm mesh layer of nylon cloth. The cells were then centrifuged at 50 · g for Abbreviations: APOBEC, apolipoprotein B mRNA-editing enzyme 2 min to collect the mature hepatocytes. The hepatocytes were then catalytic subunit; AID, activation-induced cytidine deaminase; TNF-a, suspended at a density of 5 · 105 cells/mL in Dulbecco’s modified Ea- tumour necrosis factor-a; IL-1b, interleukin-1b; NF-jB, nuclear fact- gle’s medium supplemented with 10% foetal bovine serum. The cells or-jB; IKK, IjB kinase; NEMO, NF-jB essential modulator; HCC, were seeded for 24 h to allow their attachment to the dishes, and were hepatocellular carcinoma; rRNA, ribosomal RNA; NRE, NF-jB then further treated with human TNF-a. Total RNA was extracted response element from those primary cells 12 h later.

0014-5793/$32.00 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2005.12.081 732 T. Matsumoto et al. / FEBS Letters 580 (2006) 731–735

2.2. Isolation of total RNA and quantitative RT-PCR A Total RNA was isolated using the Sepasol RNA-I (Nacalai Tesque, Kyoto, Japan) [20]. APOBEC2 expression was quantified by real-time APOBEC1 APOBEC2 APOBEC3A APOBEC3B PCR using TaqMan Universal PCR Master Mix (PE Applied Biosys- tems). The oligonucleotide primers and the 6-carboxyfluorescein 10 10 10 10 (FAM)-labelled probe used to quantify APOBEC2 amplification were 50-CCAGGCTGCTCTGAAGAAGC-30 (forward), 50-AGGCCTTG- GATTCACCCTCT-30 (reverse), and 50-TGCGCATCATGAA- 0 GCCCCAGGA-3 (probe). Eight human APOBEC family transcripts 5 5 5 5 were quantitatively amplified using the Platinum SYBR Green qPCR

SuperMix UDG (Invitrogen) with the oligonucleotide primers shown (folds of control) in Supplemental Table 1. 1 1 1 1

To quantify isolated RNA and to measure cDNA synthesis effi- Relative expression levels 0 0 0 0 ciency, target cDNAs were normalized to the expression levels of the TNF-α -+ -+ - + - + endogenous reference housekeeping gene, 18S ribosomal RNA (rRNA). The oligonucleotide primers and FAM-labelled probe used APOBEC3C APOBEC3D APOBEC3F APOBEC3G for 18S rRNA amplification and detection were 50-TAG- AGTGTTCAAAGCAGGCCC-30 (forward), 50-CCAACAAATA- 0 0 GAACCGCGGT-3 (reverse), and 5 -CGCCTGGATA- 10 10 10 10 CCGCAGCTAGGAATAATG-30 (probe). For simplicity, the expres- sion levels of the target genes were expressed as values relative to the control in each experiment. 5 5 5 5 2.3. Plasmid constructs APOBEC2 promoter fragments were generated by PCR amplifica- (folds of control) tion using Takara EX Taq DNA polymerase (Takara, Tokyo, Japan) 1 1 1 1 and the oligonucleotide primers shown in Supplemental Table 2. All Relative expression levels 0 0 0 0 reporter plasmids were constructed by inserting PCR products into TNF-α -+ -+ -+ -+ pGL3-Basic (Promega, Madison, WI). Plasmids carrying dominant negative forms of IjB kinase-a (IKK-a)orIjB kinase-b (IKK-b) BC genes, pcDNA3-IKKa (K44A) and pcDNA3-IKKb (K44A), respec- 15 10 tively, have been described previously [21].

10 2.4. Small interfering RNA To make small interfering RNA (siRNA) to target the IjB kinase-c/ 5 NF-jB essential modulator (IKK-c/NEMO), the SMARTpool kit was 5 obtained from Dharmacon Research (Lafayette, CO). siRNA was transfected into HepG2 cells using TransIT-TKO siRNA Transfection (folds of control) (folds of control) 1 1 Reagent (Mirus Bio Corporation). At 36 h after transfection, the cells APOBEC2 expression 0 APOBEC2 expression 0 were lysed for immunoblotting or were subjected to treatment with TNF-a. TNF-α -+ TNF-α -+

Fig. 1. Effects of TNF-a stimulation on the expression of APOBEC 2.5. Immunoblotting analysis and promoter assay family genes in human hepatoma cell lines. The expression of the Sample preparation and immunoblotting analysis have been de- APOBEC family in the absence or presence of TNF-a was analysed by scribed previously [20]. Antibodies directed against IKK-c/NEMO quantitative RT-PCR using lysates from Hep3B (A), HepG2 (B), or and a-tubulin were obtained from Santa Cruz Biotechnology (Santa Huh7 cells (C). Total RNA was isolated from cells 12 h after treatment Cruz, CA). To measure the promoter activity of the 50 region of APO- with TNF-a (100 ng/mL). Relative mRNA levels were measured by a BEC2, luciferase reporter constructs or a promoterless pGL3-Basic quantitative RT-PCR method with the oligonucleotide primers shown vector were individually co-transfected with pRL-TK (the internal in Supplementary Table 1. The data are the means ± S.D. values of control plasmid; Promega) into HepG2 cells. After incubation with three independent experiments. TNF-a for 24 h, luciferase activities were measured using the Dual- Luciferase Reporter Assay System (Promega) [18].

BEC2 expression in Hep3B, HepG2, and Huh7 cells, respec- 3. Results tively (Fig. 1B and C). These findings suggest that the pro- inflammatory cytokine TNF-a specifically induces human 3.1. APOBEC2 expression is induced by pro-inflammatory APOBEC2 transcription in hepatocytes. cytokine stimulation To investigate the TNF-a-mediated induction of APOBEC2 To begin, the transcripts of eight APOBEC family genes expression more precisely, we examined the time course of this were quantified in human hepatoma-derived cells. The pro- expression by incubating HepG2 cells with TNF-a. APOBEC2 inflammatory cytokine TNF-a plays a central role in hepatic mRNA was promptly induced, and the peak level was ob- inflammation, including viral induced hepatitis and alcoholic served 18 h after TNF-a treatment (Fig. 2A). Moreover, liver damage [18,22,23]. Therefore, we examined the effects TNF-a increased the amount of APOBEC2 transcript in a of TNF-a on the expression of APOBEC family genes in hepa- dose-dependent manner, when measured 12 h after the begin- tocytes. Although the transcripts of all eight APOBEC family ning of stimulation, whereas the expression of the internal con- genes examined were detectable in Hep3B cells, only APO- trol 18S rRNA gene was constant under all conditions tested BEC2 expression was markedly elevated after TNF-a treat- (Fig. 2B). To confirm the TNF-a-mediated induction of APO- ment (Fig. 1A). Similar findings were obtained using the BEC2 expression in human hepatocytes, we established cul- human hepatoma-derived cell lines HepG2 and Huh7. TNF- tured primary human hepatocytes and investigated whether a stimulation resulted in 6-, 12-, and 8-fold increases in APO- APOBEC2 expression was induced by TNF-a. Real-time T. Matsumoto et al. / FEBS Letters 580 (2006) 731–735 733

A B

8 10

6

4 5 (folds of control) (folds of control) 2 Relative APOBEC2 expression

Relative APOBEC2 expression 1 0 0 0 6 12 18 24 0 12.5 25 50 100 Time (h) Concentration (ng/ml)

C D 5 4

4 3

3 2 2 (folds of control) (folds of control) 1 1 Relative APOBEC2 expression Relative APOBEC2 expression 0 0 Fig. 3. Effects of inhibition of the NF-jB signalling pathways on TNF-α - + 0 6 12 18 APOBEC2 expression. (A) SN50 (50 lg/mL) or MG132 (2 lg/mL) was Time (h) added to HepG2 cells 1 h before stimulation with TNF-a (12.5 ng/mL). Total RNA was isolated from cells 12 h after incubation with TNF-a, Fig. 2. Dose- and time-dependent effects of TNF-a and time-depen- and expression levels of APOBEC2 were measured by real-time PCR. dent effects of IL-1b on the expression of APOBEC2. HepG2 cells were (B) pcDNA3-IKKa (K44A), pcDNA3–IKKb (K44A), or control incubated with the indicated concentrations of TNF-a for 12 h (B), or vector was transfected into HepG2 cells. Cells were treated with TNF- with 12.5 ng/mL TNF-a (A) or 5 ng/mL IL-1b (D) for the indicated a 24 h after plasmid transfection. The normalized expression levels of times. (C) Changes in APOBEC2 expression after treatment with APOBEC2 were determined by quantitative real-time PCR. (C) TNF-a (12.5 ng/mL) in cultured primary human hepatocytes. In each HepG2 cells were transfected with siRNA targeting IKK-c/NEMO experiment, total RNA was isolated after cytokine stimulation, and or control RNA, and 24 h after transfection the cell lysates were real-time PCR was performed using FAM-labelled probes specific for analysed by western blotting probed with anti-IKK-c/NEMO anti- human APOBEC2. The results were normalized to 18S rRNA body (upper panel) or anti-a-tubulin antibody (lower). (D) After expression. The data are the means ± S.D. values of three independent treatment with IKK-c/NEMO siRNA or control RNA for 24 h, experiments. HepG2 cells were treated with TNF-a (100 ng/mL) for an additional 12 h, and the APOBEC2 expression level in each extract was quantified by real-time PCR. CTR, control.

RT-PCR analysis with FAM-labelled probes revealed the up- regulation of APOBEC2 transcripts in primary hepatocytes 12 h after treatment with TNF-a (Fig. 2C). (IKK-a and IKK-b) and a non-catalytic subunit (IKK-c/ Next, we examined the effects of another pro-inflammatory NEMO) [24]. TNF-a-induced APOBEC2 expression was al- cytokine, IL-1b,onAPOBEC2 expression levels. Quantitative most completely abolished by co-production of the dominant analysis revealed that APOBEC2 transcripts increased 18 h negative form of IKK-a or IKK-b (Fig. 3B). after IL-1b stimulation (Fig. 2D). To clarify further the involvement of NF-jBinAPOBEC2 expression, we used siRNA to reduce the expression of endog- 3.2. Human APOBEC2 expression is regulated by NF-jB enous IKK-c/NEMO. We confirmed that transfection of Both TNF-a and IL-1b activate the transcription factor NF- IKK-c-specific siRNA, but not control siRNA, substantially jB and contribute to the expression regulation of various reduced endogenous IKK-c levels in HepG2 cells genes. Our finding that these cytokines induce APOBEC2 tran- and that the lower levels were sustained for at least three days scription prompted us to test whether APOBEC2 expression is (Fig. 3C). Under these conditions, TNF-a stimulated the mediated by an NF-jB-dependent mechanism. As expected, expression of APOBEC2 in control cells. In contrast, TNF-a pre-treatment with SN50 or MG132, inhibitors of NF-jB acti- failed to elicit an increase in APOBEC2 mRNA in cells in vation, almost completely suppressed the TNF-a-induced in- which endogenous IKK-c was reduced by siRNA (Fig. 3D). crease in APOBEC2 mRNA (Fig. 3A). Activation of the These findings show that the induction of APOBEC2 expres- classical NF-jB pathway converges on the IjB kinase signal- sion in human hepatocytes by TNF-a is achieved through some, a protein complex composed of two kinase subunits the activation of NF-jB. 734 T. Matsumoto et al. / FEBS Letters 580 (2006) 731–735

4. Discussion

APOBEC2 is a member of the cytidine deaminase superfam- ily that can edit RNA or DNA [14,15]. We have shown that APOBEC2 is induced in hepatocytes by the pro-inflammatory cytokines TNF-a and IL-1b through the activation of NF-jB, whereas the transcription of the other APOBEC family genes is not controlled by these pro-inflammatory cytokines. We also found that the putative NRE at 625/616 in the 50 flanking region of the APOBEC2 gene functions in APOBEC2 tran- scriptional control. Interestingly, recent studies have shown that the aberrant expression of molecules that edit DNA or RNA contributes to tumourigenesis. Hepatic overexpression of APOBEC1 re- sults in liver dysplasia and hepatocellular carcinoma (HCC) in transgenic animals [26]. Moreover, transgenic mice that con- tinuously express the editing enzyme AID develop malignant Fig. 4. Characterization of the APOBEC2 promoter region. (A) T-cell lymphomas, concomitant with frequent mutations in Nucleotide sequence of the 50 untranslated region of human APO- the T-cell receptor gene and c-myc gene [27]. These phenomena BEC2. The transcription start site (NM_006789) is designated +1, and can be explained by the abnormal editing of genes involved in the numbers indicate the nucleotide locations with respect to the growth, differentiation, or apoptosis. It is widely recognized transcription start site. The boxes indicate the two putative NF-jB response elements (NREs). (B) APOBEC2 promoter deletion con- that most human HCCs arise in a context of chronically in- structs and analysis of the promoter region of human APOBEC2. flamed liver with the features of liver cirrhosis or chronic hep- APOBEC2 promoter DNA sequences with various 50 deletions were atitis [28–30]. Our findings that pro-inflammatory cytokines subcloned upstream from the luciferase reporter gene in the pGL3- induce APOBEC2 expression in hepatocytes indicate that the Basic vector. The reporter plasmids containing two nucleotide alter- induction of such genes during inflammation may enhance sus- ations in the NREs at 625/616 (Mut-1: 50-CTGAGGCTCC-30 and Mut-2: 50-GGGAGGCTAA-30) were also constructed. HepG2 cells ceptibility to somatic gene alterations, in turn facilitating HCC were transiently transfected with various reporter constructs. At 24 h development. Further studies are required to identify the role after transfection, the cells were treated with TNF-a (12.5 ng/mL) for of APOBEC2 in the liver and to discover the specific substrates 24 h. Transfection efficiencies were normalized to Renilla luciferase of the enzyme. activity, and the results are represented as relative luciferase activities compared with those of non-treated cells. The data are the means ± S.D. values of three independent experiments. Acknowledgements: This study was supported by Grants-in-Aid for Scientific Research (15209024 and 16790378) from the Japan Society for the Promotion of Science. 3.3. Identification of the APOBEC2 promoter region that confers NF-jB-dependent activation Appendix A. Supplementary data Finally, we evaluated whether NF-jB response elements 0 (NREs) might be present in the DNA 5 to APOBEC2 (see Supplementary data associated with this article can be GenBank NM_006789). Examination of the 2000/+100 re- found, in the online version, at doi:10.1016/j.febs- gion of APOBEC2 identified two putative NREs at 625/ let.2005.12.081. 616 (50-GGGAGGCTCCT-30) and 81/72 (50- GGGAAAGCAC-30), which differed by one and three base pairs, respectively, from the consensus NRE (50- GGGNNTYYCC-30)(Fig. 4A) [25]. We then examined References whether these NREs are functional in hepatocytes by con- [1] Wedekind, J.E., Dance, G.S., Sowden, M.P. and Smith, H.C. structing plasmids with the APOBEC2 promoter, or mutants (2003) Messenger RNA editing in mammals: new members of the thereof, driving luciferase transcription. TNF-a stimulation APOBEC family seeking roles in the family business. Trends of HepG2 cells transfected with APOBEC2-promoter(1966/ Genet. 19, 207–216. 1)–luc induced a threefold increase in luciferase activity com- [2] Pham, P., Bransteitter, R. and Goodman, M.F. (2005) Reward versus risk: DNA cytidine deaminases triggering immunity and pared with that of the control cells (Fig. 4B). The highest TNF- disease. Biochemistry 44, 2703–2715. a-induced increase in promoter activity was observed with the [3] Cascalho, M. (2004) Advantages and disadvantages of cytidine APOBEC2-promoter(930/1)–luc construct. In contrast, . J. Immunol. 172, 6513–6518. plasmids lacking the putative NRE at 625/616 showed less [4] Teng, B., Burant, C.F. and Davidson, N.O. (1993) Molecular luciferase induction upon TNF-a treatment, whereas the con- cloning of an apolipoprotein B messenger RNA editing protein. Science 260, 1816–1819. struct lacking the putative NRE at 81/72 showed a level of [5] Navaratnam, N. et al. (1993) The p27 catalytic subunit of the luciferase induction similar to that of the APOBEC2-pro- apolipoprotein B mRNA editing enzyme is a cytidine deaminase. moter(1966/1)–luc construct. Moreover, the reporter activ- J. Biol. Chem. 268, 20709–20712. ities in cells transfected with plasmids encoding an NRE [6] Yamanaka, S., Poksay, K.S., Balestra, M.E., Zeng, G.Q. and Innerarity, T.L. (1994) Cloning and mutagenesis of the rabbit (625/616) mutated by two nucleotides were not affected ApoB mRNA editing protein. A zinc motif is essential for by TNF-a stimulation. These findings indicate that the distal catalytic activity, and noncatalytic auxiliary factor(s) of the candidate NRE (625/616) is required to activate the tran- editing complex are widely distributed. J. Biol. Chem. 269, 21725– scription of human APOBEC2. 21734. T. Matsumoto et al. / FEBS Letters 580 (2006) 731–735 735

[7] Driscoll, D.M. and Zhang, Q. (1994) Expression and character- necrosis factor alpha-mediated apoptosis via NF-kappaB activa- ization of p27, the catalytic subunit of the apolipoprotein B tion. J. Virol. 73, 4713–4720. mRNA editing enzyme. J. Biol. Chem. 269, 19843–19847. [19] Hino, H. et al. (1999) A long-term culture of human hepatocytes [8] Sheehy, A.M., Gaddis, N.C., Choi, J.D. and Malim, M.H. (2002) which show a high growth potential and express their differen- Isolation of a human gene that inhibits HIV-1 infection and is tiated phenotypes. Biochem. Biophys. Res. Commun. 256, 184– suppressed by the viral Vif protein. Nature 418, 646–650. 191. [9] Mangeat, B., Turelli, P., Caron, G., Friedli, M., Perrin, L. and [20] Iwai, A., Marusawa, H., Matsuzawa, S., Fukushima, T., Hijikata, Trono, D. (2003) Broad antiretroviral defence by human APO- M., Reed, J.C., Shimotohno, K. and Chiba, T. (2004) Siah-1L, a BEC3G through lethal editing of nascent reverse transcripts. novel transcript variant belonging to the human Siah family of Nature 424, 99–103. proteins, regulates beta-catenin activity in a p53-dependent [10] Harris, R.S., Bishop, K.N., Sheehy, A.M., Craig, H.M., Petersen- manner. Oncogene 23, 7593–7600. Mahrt, S.K., Watt, I.N., Neuberger, M.S. and Malim, M.H. [21] Marusawa, H., Hijikata, M., Watashi, K., Chiba, T. and (2003) DNA deamination mediates innate immunity to retroviral Shimotohno, K. (2001) Regulation of Fas-mediated apoptosis infection. Cell 113, 803–809. by NF-kappaB activity in human hepatocyte derived cell lines. [11] Zhang, H., Yang, B., Pomerantz, R.J., Zhang, C., Arunachalam, Microbiol. Immunol. 45, 483–489. S.C. and Gao, L. (2003) The cytidine deaminase CEM15 induces [22] Bradham, C.A., Plumpe, J., Manns, M.P., Brenner, D.A. and hypermutation in newly synthesized HIV-1 DNA. Nature 424, Trautwein, C. (1998) Mechanisms of hepatic toxicity. I. 94–98. TNF-induced liver injury. Am. J. Physiol. 275, G387– [12] Muramatsu, M., Sankaranand, V.S., Anant, S., Sugai, M., G392. Kinoshita, K., Davidson, N.O. and Honjo, T. (1999) Specific [23] Tai, D.I. et al. (2000) Activation of nuclear factor kappaB in expression of activation-induced cytidine deaminase (AID), a hepatitis C virus infection: implications for pathogenesis and novel member of the RNA-editing deaminase family in germinal hepatocarcinogenesis. Hepatology 31, 656–664. center B cells. J. Biol. Chem. 274, 18470–18476. [24] Bonizzi, G. and Karin, M. (2004) The two NF-kappaB activation [13] Muramatsu, M., Kinoshita, K., Fagarasan, S., Yamada, S., pathways and their role in innate and adaptive immunity. Trends Shinkai, Y. and Honjo, T. (2000) Class switch recombination and Immunol. 25, 280–288. hypermutation require activation-induced cytidine deaminase [25] Schmitz, M.L. and Baeuerle, P.A. (1995) Multi-step activation of (AID), a potential RNA editing enzyme. Cell 102, 553–563. NF-kappa B/Rel transcription factors. Immunobiology 193, 116– [14] Liao, W., Hong, S.H., Chan, B.H., Rudolph, F.B., Clark, S.C. 127. and Chan, L. (1999) APOBEC-2, a cardiac- and skeletal muscle- [26] Yamanaka, S., Balestra, M.E., Ferrell, L.D., Fan, J., Arnold, specific member of the cytidine deaminase supergene family. K.S., Taylor, S., Taylor, J.M. and Innerarity, T.L. (1995) Biochem. Biophys. Res. Commun. 260, 398–404. Apolipoprotein B mRNA-editing protein induces hepatocellular [15] Anant, S., Mukhopadhyay, D., Sankaranand, V., Kennedy, S., carcinoma and dysplasia in transgenic animals. Proc. Natl. Acad. Henderson, J.O. and Davidson, N.O. (2001) ARCD-1, an - Sci. USA 92, 8483–8487. 1-related cytidine deaminase, exerts a dominant negative effect on [27] Okazaki, I.M., Hiai, H., Kakazu, N., Yamada, S., Muramatsu, C to U RNA editing. Am. J. Physiol. Cell Physiol. 281, C1904– M., Kinoshita, K. and Honjo, T. (2003) Constitutive expression C1916. of AID leads to tumorigenesis. J. Exp. Med. 197, 1173– [16] Conticello, S.G., Thomas, C.J., Petersen-Mahrt, S.K. and Neu- 1181. berger, M.S. (2005) Evolution of the AID/APOBEC family of [28] Thorgeirsson, S.S. and Grisham, J.W. (2002) Molecular patho- polynucleotide (deoxy)cytidine deaminases. Mol. Biol. Evol. 22, genesis of human hepatocellular carcinoma. Nat. Genet. 31, 339– 367–377. 346. [17] Mikl, M.C., Watt, I.N., Lu, M., Reik, W., Davies, S.L., [29] Bruix, J., Boix, L., Sala, M. and Llovet, J.M. (2004) Focus on Neuberger, M.S. and Rada, C. (2005) Mice deficient in APO- hepatocellular carcinoma. Cancer Cell 5, 215–219. BEC2 and APOBEC3. Mol. Cell. Biol. 25, 7270–7277. [30] Feitelson, M.A., Sun, B., Satiroglu Tufan, N.L., Liu, J., Pan, J. [18] Marusawa, H., Hijikata, M., Chiba, T. and Shimotohno, K. and Lian, Z. (2002) Genetic mechanisms of hepatocarcinogenesis. (1999) Hepatitis C virus core protein inhibits Fas- and tumor Oncogene 21, 2593–2604.