TheStress-ResponsiveGene GADD45G IsaFunctionalTumor … · identified as an interleukin-2-induced immediate-early gene (11–13). These family proteins share 55% to 58% amino acid

The Stress-Responsive GeneGADD45G Is a Functional TumorSuppressor, with Its Response to Environmental StressesFrequently Disrupted Epigenetically inMultipleTumorsJianming Ying,1,2 Gopesh Srivastava,3 Wen-Son Hsieh,1,4 Zifen Gao,5 Paul Murray,6

Shuen-Kuei Liao,7 Richard Ambinder,4 and Qian Tao1,2,4

Abstract The CpG island of GADD45G was identified as a target sequence during the identification ofhypermethylated genes using methylation-sensitive representational difference analysis com-bined with 5-aza-2V-deoxycytidine demethylation. Located at the commonly deleted region9q22, GADD45G is a member of the DNA damage-inducible gene family. In response to stressshock, GADD45Ginhibits cell growth and induces apoptosis. Same as otherGADD45members,GADD45G is ubiquitously expressed in all normal adult and fetal tissues. However, its transcrip-tional silencing or down-regulation and promoter hypermethylation were frequently detected intumor cell lines, including 11of 13 (85%) non-Hodgkin’s lymphoma, 3 of 6 (50%) Hodgkin’slymphoma, 8 of 11 (73%) nasopharyngeal carcinoma, 2 of 4 (50%) cervical carcinoma, 5 of 17(29%) esophageal carcinoma, and 2 of 5 (40%) lung carcinoma and other cell lines but not inany immortalized normal epithelial cell line, normal tissue, or peripheral blood mononuclear cells.The silencing of GADD45G could be reversed by 5-aza-2V-deoxycytidine or genetic doubleknockout of DNMT1 and DNMT3B, indicating a direct epigenetic mechanism. Aberrant methyl-ation was further frequently detected in primary lymphomas although less frequently in primarycarcinomas. Only one single sequence change in the coding region was detected in 1of 25 celllines examined, indicating that genetic inactivation of GADD45G is very rare. GADD45G couldbe induced by heat shock or UV irradiation in unmethylated cell lines; however, this stressresponse was abolished when its promoter becomes hypermethylated. Ectopic expression ofGADD45G strongly suppressed tumor cell growth and colony formation in silenced cell lines.These results show thatGADD45G can act as a functional new-age tumor suppressor but beingfrequently inactivated epigenetically inmultiple tumors.

Epigenetic inactivation of tumor suppressor genes (TSG) isfrequently associated with tumor pathogenesis (1). The majormechanism of this epigenetic inactivation is through hyper-methylation of promoter CpG islands, which leads to thebinding of transcription repressors, compressed chromatin, and

transcription silencing (1). Increasing number of TSGs hasbeen documented with epigenetic inactivation in tumors, suchas p16INK4a, hMLH1, VHL, BRCA1 , and RASSF1A (2).Furthermore, promoter hypermethylation can be used as abiological marker for the identification of novel candidateTSGs and tumor diagnosis (3). Various methylation-basedstrategies, including methylation-sensitive representationaldifference analysis (MS-RDA; ref. 4), restriction landmarkgenome scanning (5), arbitrarily primed PCR (6), and CpGisland microarray (7), have been developed and proven to beuseful for identifying hypermethylated sequences. MS-RDAhas been successfully used to identify silenced genes intumors, imprinted regions on mouse chromosome 2, anddifferentially methylated regions after viral infections(reviewed in ref. 4).

GADD45 is a family of proteins involved in DNA damageresponse and cell growth arrest. GADD45A was initiallyidentified as a gene rapidly induced by DNA-damaging agents,such as methylmethane sulfonate, UV radiation, hydroxyurea,and ionizing radiation (8). It is a classic TP53-regulatedgene, and GADD45A-null mice exhibit a phenotype similar toTP53-null mice (9). GADD45B (MyD118) was firstly identi-fied as a myeloid differentiation responsive gene, activated inM1 myeloid leukemia cells by interleukin-6 after induction ofterminal differentiation (10). GADD45G (GRP17/CR6) was

Human Cancer Biology

Authors’Affiliations: 1Johns Hopkins Singapore, Singapore; 2Cancer EpigeneticsLaboratory, Department of Clinical Oncology, SirYK Pao Cancer Center, ChineseUniversity of Hong Kong; 3Department of Pathology, University of Hong Kong,Hong Kong; 4Sydney Kimmel Comprehensive Cancer Center, Johns HopkinsSchool of Medicine, Baltimore, Maryland; 5Department of Pathology, PekingUniversity Health Science Center, Beijing, China; 6Cancer Research UK Institutefor Cancer Studies, University of Birmingham, Birmingham, United Kingdom;and 7Graduate Institute of Clinical Medical Sciences, Chang Gung University,Taiwan, ChinaReceived 2/4/05; revised 4/4/05; accepted 4/13/05.Grant support: A*STAR, Singapore, Chinese University of Hong Kong, andLymphoma Specialized Programs of Research Excellence grant P50CA96888(R. Ambinder).The costs of publication of this article were defrayed in part by the payment of pagecharges.This article must therefore be hereby marked advertisement in accordancewith18 U.S.C. Section1734 solely to indicate this fact.Requests for reprints: Qian Tao, Department of Clinical Oncology, Sir YK PaoCancer Center, Prince of Wales Hospital, Chinese University of Hong Kong,Room 315, Hong Kong. Phone: 852-2632-1340; Fax: 852-2648-8842; E-mail:[email protected].

F2005 American Association for Cancer Research.doi:10.1158/1078-0432.CCR-05-0267

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identified as an interleukin-2-induced immediate-early gene(11–13). These family proteins share 55% to 58% amino acidhomology and mediate the activation of the p38/c-Jun NH2-terminal kinase pathway via MTK1/MEKK4 in response tovarious environmental stresses (11). They can also inhibitcell proliferation at different stages, including G1-S and G2-Mcheckpoints, and induce cell apoptosis (11, 13–15). GADD45Bis down-regulated in hepatocellular carcinoma through methy-lation (16), and GADD45G is also down-regulated in anaplasticthyroid cancer and pituitary adenoma (17, 18).

To identify aberrantly methylated genes, we used a novelapproach by combining MS-RDA with demethylation treatmentin cell lines of nasopharyngeal carcinoma, a prevalent tumor inour locality of Southern China and Southeast Asia. Weidentified GADD45G as a target gene for epigenetic inactivationin nasopharyngeal carcinoma as well as multiple other types ofcarcinomas and lymphomas. We further showed that exoge-nous expression of GADD45G strongly suppressed tumor cellgrowth and colony formation, indicating that GADD45G canact as a functional TSG.

Materials andMethods

Cell lines and primary tumors. In total, 75 cell lines of carcinomasand lymphomas were used, including nasopharyngeal (C666-1, CNE-1,CNE-2, HK1, NPC-BM1, HNE1, HNE2, HNE3, HONE-1, 5-8F, andHKM1), breast (MCF-7, T47D, ZR-75-1, and MDA-MB231), esophageal(EC1, EC18, EC109, HKESC-1, HKESC-2, HKESC-3, SLMT-1, KYSE30,

KYSE70, KYSE140, KYSE150, KYSE180, KYSE270, KYSE410, KYSE450,KYSE510, and KYSE520), colorectal (HCT116, HT-29, HCT15, SW48,and LoVo), cervical (HeLa, CaSki, C33A, and SiHa), lung (A549,H1299, H2126, H1395, and H292), hepatocellular (HepG2, huH1,huH4, huH6, and huH7), gastric (KatoIII), and laryngeal carcinoma(HEp-2), non-Hodgkin’s lymphoma (BJAB, CA46, Rael, Namalwa, Raji,AG876, OCI-Ly1, Ly3, Ly7, Ly8, Ly13.2, Ly17, and Ly18), Hodgkin’slymphoma (L428, L540, L591, HD-LM2, HD-MY-Z, and KM-H2), andleukemia (HL-60, THP-1, and K562). Four immortalized normalepithelial cell lines (NP69, NE1, NE3, and RHEK-1; refs. 19, 20) withmany features of normal epithelial cells were also used. HCT116DNMT1�/� (1KO), HCT116 DNMT3B�/� (3BKO), and HCT116DNMT1�/�DNMT3B�/� (DKO) cells (gifts of Bert Vogelstein, JohnsHopkins, Baltimore, MD) were grown with either 0.4 mg/mL geneticinor 0.05 mg/mL hygromycin (21). DNA and RNA of primarycarcinomas and lymphomas have been described (19, 22–25). Celllines were treated with 5-aza-2V-deoxycytidine (Sigma, St. Louis, MO) aspreviously (19). For the treatment combining 5-aza-2V-deoxycytidineand trichostatin A (Cayman Chemical Co., Ann Arbor, MI), cells weretreated with 5-aza-2V-deoxycytidine for 3 days and subsequently withtrichostatin A (100 ng/mL) for 24 hours.

Stress treatments. Heat shock was done as previously (19), exceptfor an incubation at 42jC for 1 hour with recovery at 37jC for2 hours. For UV treatment, medium was removed and the flask wasturned upside down to face the light source in a UV cross-linker 500(Amersham Biosciences, Piscataway, NJ). Cells were irradiated for adose of 70 J/m2. After irradiation, fresh medium was added, and thecells were recovered at 37jC for 1 hour and then harvested.

Methylation-sensitive representational difference analysis. MS-RDAwas done as previously (26). We used the genomic DNA of a

Table1. Sequences of primers used in this study

PCR Primers Size (bp) Annealingtemperature (�C)

Cycles

RT-PCRGADD45A F: 5V-TGAGTGAGTGCAGAAAGCAG 181 55 35, 37

R: 5V-TTTGCTGAGCACTTCCTCCAGADD45B F: 5V-AACATGACGCTGGAAGAGCT 247 55 35, 37

R: 5V-AGAAGGACTGGATGAGCGTGGADD45G F: 5V-AACTAGCTGCTGGTTGATCG 178 55 35, 37

R1: 5V-CGTTCAAGACTTTGGCTGACR2 (for direct sequencing and cloning):5V-ACCACGTCGATCAGACCAAG

539 55 32

GAPDH 33: 5V-GATGACCTTGCCCACAGCCT 304 60 2555: 5V-ATCTCTGCCCCCTCTGCTGA

MSP for theGADD45B promoterGADD45B m3: 5V-GAAAGTTCGGGTCGTTTCGC 134 60 40

m4: 5V-GAAAACCGAATAAATAACCGCGu3: 5V-TTTGAAAGTTTGGGTTGTTTTGT 139 58 40u4: 5V-ACAAAAACCAAATAAATAACCACA

MSP for theGADD45G promoterRegion1 (top strand) m1: 5V-ACGTGGTTTTTTGGTACGAGTC 160 64 40

m2: 5V-GCCCACCACCAACGAATACGu1: 5V-ATGTGGTTTTTTGGTATGAGTT 160 58 40u2: 5V-ACCCACCACCAACAAATACA

Region 2 (bottom strand) bm1: 5V-CGGAATTGTGTTTTGGTCGC 185 58 40bm2: 5V-ACCAACCTATATAAAAACGCGbu1: 5V-TTTGGAATTGTGTTTTGGTTGT 188 58 40bu2: 5V-CACCAACCTATATAAAAACACA

BGS for theGADD45G promoter BGS1: 5V-GTAGATTTGAGGTATTGTTATTT 355 56 40BGS2: 5V-CCTAAAACCCACCTAACTATA

Epigenetic Silencing of GADD45G inMultiple Tumors

www.aacrjournals.org Clin Cancer Res 2005;11(18) September15, 20056443



nasopharyngeal carcinoma cell line CNE-1 (the Driver) and CNE-1treated with 50 Amol/L 5-aza-2V-deoxycytidine (the Tester) and did twocycles of competitive hybridization. PCR products from the secondhybridization were cloned into the pCR2.1-Topo vector (Invitrogen,Carlsbad, CA), sequenced, and analyzed using the BLASTN algorithm(National Center for Biotechnology Information).

5V-Rapid Amplification of cDNA Ends. We determined the GADD45Gtranscription start site using 5V-Rapid Amplification of cDNA Endsversion 2.0 (Invitrogen). Briefly, the first-strand cDNA was synthesizedfrom placenta RNA using primer GADD45GR2 (Table 1). Homopol-ymeric tails were then added to the 3V ends with terminal deoxy-nucleotidyl transferase. PCR was done using Abridged Anchor Primerand a second gene-specific primer GADD45GR1 (Table 1). The RapidAmplification of cDNA Ends product was enriched by reamplifyingwith the Abridged Universal Amplification Primer and GADD45GR1,cloned, and sequenced.

Semiquantitative reverse transcription-PCR analysis. Reverse tran-scription-PCR (RT-PCR) was done as previously (22, 27). Primers areshown in Table 1.

Bisulfite treatment and promoter methylation analysis. Bisulfatemodification of DNA, methylation-specific PCR (MSP), and bisulfitegenomic sequencing (BGS) were carried out as previously (27, 28).GADD45B promoter and two regions of the GADD45G promoter wereanalyzed by MSP (Table 1). MSP primers were tested previously for notamplifying any unbisulfited DNA.

Analyses of tumor suppressor functions. The full-length cDNA ofGADD45G was PCR cloned from human trachea RNA (BD Clontech,Palo Alto, CA), sequence verified, and then subcloned into bothpcDNA3.1(+) and pcDNA3.1(�) vectors (Invitrogen) to generate senseplasmid pcDNA3.1(+)GADD45G and antisense plasmid pcDNA3.1(�)GADD45G. For colony formation assay using monolayer culture, cells(2 � 105 per well) were plated in a 12-well plate and transfected with2 Ag sense, antisense, or vector plasmid using LipofectAMINE 2000(Invitrogen). Cells were stripped off, plated in a six-well plate 48hours post-transfection, and selected for 2 to 3 weeks with 0.4 to 0.5mg/mL G418. Surviving colonies (z50 cells per colony) were countedafter staining with Giemsa. For colony formation assay using soft agarculture, cells were transfected as above and suspended in RPMI 1640containing 0.35% agar, 10% fetal bovine serum, and G418 andthen layered on RPMI 1640 containing 0.5% agar, 10% fetal bovineserum, and G418 in a six-well plate 48 hours post-transfection.Colonies were counted and photographed 16 days post-transfection.Total RNA from transfected cells was extracted, treated with DNase I,and analyzed by RT-PCR to confirm the ectopic expression ofGADD45G . All experiments were done in triplicate wells and repeatedthrice.

Cell proliferation assay. CA46 cells (1 � 106 per well in 12-wellplate) were transfected with empty vector or pcDNA3.1(+)GADD45Gand plated in a 12-well plate (7.5 � 104 per well) 12 hours post-transfection. Cells were counted at indicated time points using a

Fig. 1. Schematic structure of theGADD45G CpG island. A, locations of thehypermethylated fragment identified byMS-RDA, the core promoter, exons1to 3,and part of exon 4 (filled rectangles).Thetranscription start site mapped by 5V-RapidAmplificationof cDNAEnds is indicatedby abent arrow. Six heat shock factorswithin thecore promoter are also shown.TwoMSPregions and the BGS region analyzed areindicated. B, expression profiling ofGADD45A, GADD45B, andGADD45G inhumannormal adult and fetal tissues bysemiquantitative RT-PCR, withGAPDH as acontrol. S.M., skeletonmuscle; B.M., bonemarrow. C, representative expression ofGADD45A, GADD45B, andGADD45Gby semiquantitative RT-PCR (top three)andmethylation status of theGADD45BandGADD45G promoters by MSP(bottom four) in various tumor cell lines.M, methylated; U, unmethylated.D, analysisofGADD45G cDNA sequence variations bydirect sequencing. +, with sequencevariation;�, no variation.





hemocytometer based on trypan blue exclusion. The experiment wasdone in triplicate wells and repeated thrice.

Screening for GADD45G mutations. Single-stranded cDNA wassynthesized with SuperScript II reverse transcriptase (Invitrogen) usingtotal RNA from cell lines or cell lines treated with 5-aza-2V-deoxycytidine. RT-PCR was used to amplify the full-length codingsequence with Accume Pfx DNA polymerase (Invitrogen) and primersGADD45GF and GADD45GR2. PCR products were electrophoresed,purified, and sequenced in both directions.

Statistical analysis. Statistical significance of the assays was analyzedby t test. P < 0.05 was taken as statistically significant.

Results and Discussion

Identification of the CpG island of GADD45G as a hyper-methylated target. By combining MS-RDA with pharmacologicdemethylation, DNA fragments hypermethylated in CNE-1 butdemethylated after 5-aza-2V-deoxycytidine treatment were iden-tified, cloned, and analyzed by bioinformatics. All but 2 of the31 unique fragments identified have typical CpG islands.8

Among them, a 296-bp fragment located at 9q22, a locusfrequently deleted in multiple tumors (29), contains part of theexon 3, exon 4, and intron 3 of the GADD45G gene (ref. 11;Fig. 1A).

Frequent epigenetic silencing of GADD45G but not GADD45Bin various cell lines. We then examined the expression ofGADD45G , together with other family members GADD45Aand GADD45B , in a series of cell lines to determine whetherthere are widespread epigenetic inactivation of these genes intumors. We analyzed 11 nasopharyngeal carcinoma and 56other cell lines using semiquantitative RT-PCR. We found thatGADD45G expression was remarkably reduced or silenced in8 of 11 nasopharyngeal carcinoma, 11 of 13 non-Hodgkin’slymphoma, 3 of 6 Hodgkin’s lymphoma, and 13 of 37 othercell lines. In contrast, the expression levels of GADD45A andGADD45B remained high in virtually all cell lines, withGADD45B the highest (Fig. 1C). Furthermore, along withGADD45A and GADD45B, GADD45G was readily detected inall 30 normal adult and fetal tissues, including normalperipheral blood mononuclear cells (Fig. 1B).

Before we could assess the epigenetic alterations ofGADD45G , we determined the accurate location of thepromoter and the transcription start site using 5V-RapidAmplification of cDNA Ends. We obtained a strong PCR bandof f260 nucleotides (data not shown). Sequence analysis ofthe product (AY845250) showed that the identified transcrip-tion start site matched exactly the published cDNA 5Vsequence(NM_006705) in National Center for Biotechnology Informa-tion database. The GADD45G promoter is located within atypical CpG island (19), containing the core promoter, exons 1to 3, and part of exon 4 (Fig. 1A). CpG islands are frequentlysilenced by methylation in tumors as an alternative epigeneticmechanism to inactivate TSG functions (1). We suspected thatthe silencing of GADD45G might also be mediated throughepigenetic regulation. Therefore, the methylation status of tworegions in the GADD45G promoter was analyzed by MSP in atotal of 75 cell lines (Fig. 1C; Table 2). The MSP results ofregion 2 were identical to that of region 1 (data not shown). Intotal, GADD45G methylation was detected in 85% (11 of 13)

non-Hodgkin’s lymphoma, 50% (3 of 6) Hodgkin’s lympho-ma, 73% (8 of 11) nasopharyngeal carcinoma, 29% (5 of 17)esophageal carcinoma, 50% (2 of 4) cervical carcinoma, and40% (2 of 5) lung carcinoma and other tumor cell lines. All thecell lines with hypermethylation had either reduced or silencedexpression depending on the extent of methylation level. Nomethylation was found in the four immortalized normalepithelial cell lines, which had virtually normal phenotypesand expressed this gene (Fig. 2A). In contrast, no methylationof the GADD45B promoter was detected, which correlated withits broad expression in cell lines (Fig. 1C).

To further examine GADD45G methylation in more detail,we analyzed 12 cell lines, 2 normal tissues, and 5 tumors usingthe high-resolution BGS analysis (Fig. 2B). We analyzed 33CpG sites spanning the core promoter and part of exon 1 in a355-bp region. The BGS results strongly correlated with theMSP analysis. Methylated CpG sites were not found or only

Table 2. Frequencies ofGADD45Gmethylation incelllines and primary tumors

Samples Promotermethylation (%)

Tumor cell linesNon-Hodgkin’s lymphoma 11/13 (85)Hodgkin’s lymphoma 3/6 (50)Leukemia 1/3 (33)Nasopharyngeal carcinoma 8/11 (73)Cervical carcinoma 2/4 (50)Lung carcinoma 2/5 (40)Esophageal carcinoma 5/17 (29)Hepatocellular carcinoma 1/5 (20)Colorectal carcinoma 1/5 (20)Laryngeal carcinoma 1/1Breast carcinoma 0/4Gastric carcinoma 0/1

Primary lymphomasEndemic Burkitt’s lymphoma 7/8 (88)Diffuse large B-cell lymphoma 5/13 (38)Follicular lymphoma 1/6 (16)Post-transplant lymphoma 4/13 (33)Nasal NK/Tcell lymphoma 5/8 (63)Hodgkin’s lymphoma 10/29 (34)Other types of lymphoma 0/11

Primary carcinomasNasopharyngeal carcinoma 6/38 (16)Esophageal carcinoma 3/27 (11)Breast carcinoma 0/20Gastric carcinoma 2/19 (11)Hepatocellular carcinoma 0/6

Immortalized normal epithelial cell linesRHEK-1, NE1, NE3, NP69 0/4

Normal tissuesPeripheral bloodmononuclear cell 0/12Lymphnode 0/3Nasopharynx 0/10Breast tissue 0/7Esophageal epithelium 0/7

8 J.Ying and Q.Tao, in preparation.





scattered in cell lines expressing GADD45G . In contrast,densely methylated CpG sites were detected in all silencedcell lines (Fig. 2B). Our data indicate that epigenetic silencingof GADD45G is involved in the pathogenesis of a fewtumors.

Activation of GADD45G expression by pharmacologic andgenetic demethylation. To test whether methylation is directlyresponsible for silencing GADD45G , we treated five cell lines(Rael, Namalwa, Raji, L428, and EC109) with hypermethylatedand silenced promoter with the demethylating agent 5-aza-2V-deoxycytidine. 5-Aza-2V-deoxycytidine restored GADD45Gexpression in all of them although with different efficiency(Fig. 3A). No significant increase of either GADD45A orGADD45B expression was detected (data not shown). Con-comitantly, after 5-aza-2V-deoxycytidine treatment, unmethy-lated GADD45G alleles were increased as determined by bothMSP and BGS (Figs. 2B and 3A). The Rael cell line showedmarginal restoration of GADD45G after 5-aza-2V-deoxycytidinetreatment. However, further treatment with 5-aza-2V-deoxycyti-dine combined with histone deacetylase inhibitor, trichostatinA, resulted in synergistic activation of GADD45G to greaterlevels (data not shown), suggesting that histone deacetylationis also involved in repressing GADD45G in this cell line.

GADD45G could also be activated to similar extent as the5-aza-2V-deoxycytidine–treated in HCT116, which has a minor-ity of methylated alleles, by genetic biallelic disruption of bothDNMT1 and DNMT3B (DKO) but not DNMT1 or DNMT3Balone (ref. 21; data not shown). In contrast, neither GADD45Anor GADD45B expression was affected in the DKO cellline (data not shown). These results indicate that themaintenance of GADD45G promoter methylation is mediatedby DNMT1 and DNMT3B together, like other known typicalTSGs (30).9

GADD45G methylation is tumor specific. We further inves-tigated the presence of GADD45G methylation in a largecollection of primary tumors, including various lymphomasand carcinomas and normal tissues (Fig. 2A and B; Table 2).GADD45G methylation was detected in multiple tumor typeswith different frequencies, more frequently in lymphomas thancarcinomas and in carcinoma cell lines than primary carcinomas(73% versus 16% in nasopharyngeal carcinoma and 29% versus11% in esophageal carcinoma; Table 2), indicating that some cell

Fig. 2. Analyses of the methylation statusofGADD45G in primary tumors.A, representative MSPanalysis ofGADD45G in primary tumors, normaltissues, and immortalized normal cell lines.PCR bands marked with M indicatemethylated promoter, whereas PCR bandsmarked with U indicate unmethylatedpromoter. B, high-resolutionmapping of themethylation status of every CpG site in theGADD45G promoter by BGS in normaltissues, tumor cell lines, primary tumors,and cell lines treated with 5-aza-2V-deoxycytidine. A 355-bp region spanningthe core promoter with 33 CpG sites wasanalyzed.Transcription start site is labeled asa curved arrow. Each CpG site is shown atthe top row as an individual number (#).Percentage methylationwas determined aspercentage of methylated cytosines from8 to10 sequenced colonies. On the rightside is theMSP results andGADD45Gexpression levels. M, methylated;U, unmethylated; (M), weakly methylated;(U), weakly unmethylated; NA, notavailable; LN, lymph node; GC, gastriccarcinoma; BL, Burkitt’s lymphoma; DLBCL,diffuse large B-cell lymphoma; PTLD,post-transplant lymphoma.

9 G.H. Qiu and Q.Tao, submitted for publication.





lines may have acquired GADD45G methylation during theestablishment or maintenance process. Similar phenomenonhas been reported for some TSGs in other tumors (31, 32). Noneof the 39 normal tissues (12 peripheral blood mononuclear cells,3 lymph nodes, 10 nasopharynx, 7 normal breast tissues, and7 normal esophageal epithelia) had methylation as assessed byeither BGS or MSP. Therefore, hypermethylation of theGADD45G promoter is tumor specific.

Genetic inactivation of GADD45G is very rare. Because TSGscan also be inactivated genetically by various mutations,including deletions, and GADD45G is located in a frequentlydeleted locus, we tested this possibility for GADD45G and didnot detect its deletion in any cell line. We further used directsequencing to screen for any possible point mutation in 25 celllines, including expressing cell lines and silenced cell linestreated with 5-aza-2V-deoxycytidine. Only a single sequencechange G344A (G112E) in exon 3 was identified in one cell lineAG876 (Figs. 1D and 3B), in which the promoter was alsomethylated. This sequence substitution might be a raremutation or a polymorphism. Therefore, the disruption ofGADD45G functions in tumor cell lines is solely throughepigenetic rather than genetic mechanism.

Promoter hypermethylation disrupts the stress response ofGADD45G. Because GADD45 family proteins are involvedin cellular responses to environmental stresses, we inspectedthe GADD45G promoter for potential stress-responsive ele-ments. Multiple heat shock factor binding sites (6 in the corepromoter, with a total of 26 sites in a region from �1,500to +150) are present in the promoter (Fig. 1A), indicating thatit is likely stress responsive. It has been reported that thispromoter is inducible by g-ray, H2O2, and UV irradiation (11).We further tested whether it is responsive to heat shock and

whether its stress response would be affected by promotermethylation. Stress treatments (heat shock and UV irradiation)of normal and tumor cell lines with an unmethylated or onlypartially methylated promoter resulted in increased GADD45Gexpression. However, this response was decreased or abolishedin cell lines with methylated promoter and was inverselycorrelated with the promoter methylation levels (Fig. 3C).Under our conditions of stress treatment (heat shock at42jC for 1 hour with 2-hour recovery and 70 J/m2 UVCtreatment with 1-hour recovery), we only observed marginalup-regulation of GADD45A but not GADD45B RNA. It hasbeen shown that different GADD45 genes respond to stressstimuli differently (i.e., GADD45A is inducible by p53, whereasGADD45B and GADD45G are not, and interleukin-12 caninduce GADD45B but not GADD45G ; ref. 15). Interestingly,in treated normal (NE3) and some tumor cell lines (EC109and Rael), two new isoforms of GADD45B were induced afterheat shock (Fig. 3C). These isoforms have extended exon 1 ofdifferent lengths, resulting in the translation of a shorterisoform (15 amino acids less) from an alternate ATG codemore downstream (accession nos. AY615270 and AY615271).The functions of these new GADD45B isoforms need furtherstudy.

GADD45G is a functional tumor suppressor. GADD45G isassociated with proliferating cell nuclear antigen and thecyclin-dependent kinase inhibitor p21WAF1/CIP1 and is involvedin the negative control of cell growth (13, 15). GADD45G isalso associated with MTK1/MEKK4, which in turn activates thep38/c-Jun NH2-terminal kinase pathway leading to apoptosis,in response to environmental stresses (11). Transient transfec-tion of GADD45 members could induce apoptosis in tumorcells (11, 13, 15). The frequent epigenetic silencing of

Fig. 3. A, pharmacologic demethylationand induction of GADD45G in methylatedand silenced tumor cell lines by 5-aza-2V-deoxycytidine. +, 5-aza-2V-deoxycytidinetreated; �, untreated. B, only a singlesequence change G443A (G112E) in thecoding region of GADD45G was identifiedin 1cell line, AG876, of 25 tumor cell linesanalyzed. C, up-regulation of GADD45G, inresponse to stress treatment, is abolished intumor cell lines with methylated promoter.Subconfluent normal (NE3) and tumorcell lines (CNE-2, HK1, EC109, and Rael)were exposed to 42jC heat shock or UVirradiation and then analyzed for GADD45A,GADD45B, and GADD45G expression.GADD45G methylation status in each cellline is shown (bottom), with percentage ofmethylated alleles indicated. The twonovel heat shock ^ induced isoforms ofGADD45B are indicated by asterisks. +,stress treated; �, mock control.





GADD45G in tumors and cell lines but not normal tissuessuggests that it might be a functional tumor suppressor. Totest this hypothesis, we cloned the full-length GADD45G cDNAinto expression vectors and transfected it into several carci-noma and lymphoma cell lines, which had either completemethylation and silencing (HepG2, EC109, HEp-2, and CA46)or only weak expression with both methylated and unmethy-lated alleles (CNE-1 and HK1). The colony formationefficiencies of each transfected cell line were evaluated bymonolayer and soft-agar culture with G418 selection. Ectopicexpression of GADD45G dramatically reduced the colonyformation efficiencies of these cell lines in both monolayercultures (down to 9-20% of controls in methylated cell linesand 19-41% in hemimethylated cell lines; Fig. 4A) and soft-agar assays (down to 8% of controls in CNE-1 and HepG2;Fig. 4B), suggesting that GADD45G can function as a bona fideTSG. Interestingly, HK1 has the relatively highest endoge-nous expression and lowest methylation level. The inhibitionof colony formation by ectopic GADD45G expression in HK1was also the least effective. Furthermore, cell proliferationassay was done for CA46 (Fig. 4C). Cells transfected withpcDNA3.1(+)GADD45G grew significantly slower than themock-transfected cells (P < 0.001), indicating that GADD45Gdramatically inhibits not only tumor cell colony formation butalso their proliferation. Previously, it has been shown thatectopic GADD45G expression in lung carcinoma, pituitarytumor, and anaplastic thyroid carcinoma–derived cell linesresulted in a substantial inhibition of tumor cell growth(17, 18). As shown in this study, GADD45G also strongly

suppressed tumor cell growth and colony formation of othertumor cell lines, including nasopharyngeal, hepatocellular,esophageal, and laryngeal carcinoma and lymphoma cell lines,further indicating that GADD45G can function as a TSG inmultiple tumors.

In summary, we identified the GADD45G CpG island as atumor-specific, hypermethylated target sequence. We alsofound that GADD45G expression is frequently reduced orsilenced in multiple tumors, including lymphomas andcarcinomas, like other class II TSGs (33). This silencing isdue to the hypermethylation of its promoter, which furtherimpairs its response to environmental stresses, but geneticinactivation of GADD45G is rare. We further showed thatGADD45G can act as a functional TSG in multiple tumorcells. During the preparation of this article, Bahar et al. alsoreported the epigenetic down-regulation of GADD45G inpituitary adenoma (34). This frequent inactivation ofGADD45G in various tumors is consistent with its proposedrole as a negative regulator of cell proliferation (13, 15) and alsoshows the importance of GADD45G in preventing thedevelopment of multiple tumors. As promoter hypermethyla-tion is pharmacologically reversible using demethylating agents,and GADD45G is rarely mutated in tumors, it is therefore alikely target for new epigenetic anticancer therapeutics.

GenBank accession numbers. The sequences of the 5V-RapidAmplification of cDNA Ends product of GADD45G (accessionno. AY845250) and heat shock–induced GADD45B isoforms(accession nos. AY615270 and AY615271) have been depositedto Genbank.

Fig. 4. Tumor suppressor function analyses ofGADD45G inmultiple tumor cell lines.A, representative inhibition of colony formation inmonolayer culture byGADD45G. HepG2 cells weretransfected with pcDNA3.1(+)GADD45G,antisense plasmid (AS), or control vector andselected with G418 for 3 weeks. Quantitativeanalyses of colony numbers in several cell lines areshown on the right .The numbers of G418-resistantcolonies in each control vector-transfected cell linewere set to100%. Columns, mean of at least threeseparate experiments; bars, SE.B, representativeinhibition of colony formation byGADD45G in softagar culture. HepG2 cells were transfected withpcDNA3.1(+)GADD45G or empty vector andselected in soft agar with G418 for16 days.Quantitative analyses of colony numbers aftertransfection and G418 selection in CNE-1andHepG2 are shown on the right. In each case,the numbers of G418-resistant colonies invector-transfected cell lines were set to100%.Columns, mean of at least three separateexperiments; bars, SE. C, growth curvesof CA46 cells after transfection withpcDNA3.1(+)GADD45G or control vector.At each indicated time point after transfection,cell numbers were counted and plotted.Points, mean of triplicate experiments;bars, SE. ***, P < 0.001.





NoteAdded in Proof

During the final preparation of this article, Zerbini et al. reported that nuclearfactor-nB ^ mediated repression of GADD45A and GADD45G is essential for can-cer cell survival (Zerbini LF, et al. Proc Natl Acad Sci U S A 2004;101:13618^23),thus pointing out another mechanism to inactiveGADD45G functions in tumors.

Acknowledgments

We thank Drs. Bert Vogelstein, Soh-ha Chan, Riccardo Dalla-Favera, MeehardHerlyn, Dolly Huang, Katai Yao, Ya Cao, Thomas Putti, Guiyuan Li, Yixin Zeng,Sen-TienTsai, SaiWahTsao, Johng S. Rhim, Malini Olivo, Goh Boon Cher, and LeeSoo Chin for some cell lines and samples.



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2005;11:6442-6449. Clin Cancer Res Jianming Ying, Gopesh Srivastava, Wen-Son Hsieh, et al. TumorsStresses Frequently Disrupted Epigenetically in MultipleTumor Suppressor, with Its Response to Environmental

Is a FunctionalGADD45GThe Stress-Responsive Gene

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TheStress-ResponsiveGene GADD45G IsaFunctionalTumor … · identified as an interleukin-2-induced immediate-early gene (11–13). These family proteins share 55% to 58% amino acid