Cloning a Novel Member of the Human Interferon-Inducible Gene Family Associated with Control of Tumorigenicity in a Model of Human Melanoma

Cloning a novel member of the human interferon-inducible gene family associated with control of tumorigenicity in a model of human melanoma

Katherine L DeYoung,1,4 Michael E Ray,1,2 Yan A Su,1 Sarah L Anzick,1 Ricky W Johnstone,3 Joseph A Trapani,3 Paul S Meltzer1 and Jerey M Trent1

1Laboratory of Cancer Genetics, National Human Genome Research Institute, National Institutes of Health, 9000 Rockville Pike, Building 49, Room 4A22, Bethesda, Maryland, 20892, USA; 2Department of Human Genetics, The University of Michigan Medical School, Medical Science II M4708, Ann Arbor, Michigan, 48109, USA; 3The Austin Research Institute, Austin Hospital, Heidelberg, 3084, Australia

Chromosome 6-mediated suppression of tumorigenicity in chromosome 1 (Trent et al., 1989; Fountain et al., 1990; malignant melanoma cell lines provides a model system Thompson et al., 1995), complete or partial loss of the to identify genes associated with the reversion of the long arm of chromosome 6 (Trent et al., 1989; Fountain tumorigenic phenotype. Using subtractive cDNA selec- et al., 1990) frequent alterations of the short arm of tion, we recently identi®ed a series of novel genes which chromosome 9 (Skolnick et al., 1994; Thompson et al., are dierentially expressed in association with chromo- 1995; Kraehn et al., 1995) and 1p11-q22 (Thompson et some 6-mediated suppression. We now report the al., 1995; Kraehn et al., 1995). Evidence of a putative molecular characterization of a novel gene termed tumor suppressor gene on chromosome 6 has been AIM2 for (Absent In Melanoma), which represents a suggested by cytogenetic observation (Trent et al., 1989; 1485 bp cDNA. An open reading frame of 1032 base Fountain et al., 1990), by high frequency of loss of pairs, corresponding to 344 amino acid residues, is heterozygosity (LOH) along 6q (Milliken et al., 1991; predicted. The predicted protein shares a conserved Walker et al., 1994) and by studies suggesting that either sequence domain of approximately 200 amino acids with tumorigenicity or metastasis of melanoma cell lines can known interferon-inducible genes of both human and be suppressed by the introduction (following micro-cell mouse. We demonstrate that the AIM2 gene encodes a mediated chromosome transfer) of a normal copy of transcript of approximately 2 kb which is expressed in human chromosome 6 (Trent et al., 1990; You et al., spleen, small intestine, and peripheral blood leukocytes. 1995; Ray et al., 1996). In addition, we have localized AIM2 to the long arm of Recently, we reported the use of a PCR-based human chromosome 1 (band q22) in a highly conserved cDNA subtractive hybridization technique to isolate a region which also contains the known interferon-inducible series of novel cDNAs which were expressed exclu- genes IFI16 and MNDA. We have also demonstrated sively in a chromosome-suppressed melanoma cell line that, like IFI16 and MNDA, AIM2 is induced in HL60 (Ray et al., 1996). This study documents the molecular cells by interferon gamma. Our ®ndings support the characterization of one of these genes, termed AIM2 existence of a family of genes in this region similar to (for Absent In Melanoma, gene #2), which was shown the well-characterized mouse I®200 gene family. to belong to an established family of interferon- inducible genes located on human chromosome 1. Keywords: interferon-inducible; melanoma; tumorigeni- AIM2 bears striking sequence homology at both the city DNA and protein levels with two previously recognized members of this gene family, IFI16 and MNDA (Trapani et al., 1992; Briggs et al., 1994), as well as with several members of the murine I®200 cluster of Introduction interferon-inducible genes (Choubey et al., 1989). In this report we discuss the isolation of a full length The transformation of normal melanocytes to malignant cDNA for AIM2, containing elements which are highly melanoma cells is thought to result from an accumula- conserved among known interferon-inducible genes, as tion of genetic alterations, although the speci®c genes well as providing preliminary examination of the involved remain largely unknown. Cytogenetic studies expression pattern of this gene in various normal have con®rmed the presence of recurring chromosomal human tissues, melanoma cell lines, and normal alterations which pinpoint the locations of growth melanocytes. We also demonstrate that AIM2 expres- regulating genes that may be involved in the progression sion is induced by interferon gamma in HL60 cells. of human malignant melanoma (Trent et al., 1989; Our results suggest that AIM2 is a novel member of a Fountain et al., 1990; Thompson et al., 1995; Kraehn et family of closely related interferon-inducible genes, al., 1995). Chromosome alterations in malignant located on human chromosome 1, which is similar to melanoma most frequently include a variety of non- the I®200 gene family in the mouse. reciprocal translocations and simple deletions involving

Results Correspondence: JM Trent 4 Present address: University of California, Davis, Briggs Hall, Davis, Sequence analysis CA 95616 Received 20 November 1996; revised 7 April 1997; accepted 17 April Analysis of 15 overlapping AIM2 cDNA clones 1997 allowed the construction of a consensus representing Cloning of a novel interferon inducible gene KL DeYoung et al 454 approximately 1485 bp of unique sequence which Chromosomal localization included the entire AIM2 coding region. Figure 1 shows the nucleotide sequence of AIM2 with the A P1 clone positively identi®ed by PCR using deduced amino acid sequence of the longest open sequence-speci®c AIM2 primers was used as a FISH reading frame noted below. The molecular weight of probe to provide sublocalization of the previously the predicted polypeptide is 39 487 Da, and the reported somatic cell hybrid mapping to chromosome 1 estimated isoelectric point is 9.76. The sequence (Ray et al., 1996). Figure 3 demonstrates the results of contains a tyrosine kinase phosphorylation site at FISH to DAPI-banded normal metaphase chromo- amino acid positions 85 ± 92. somes, allowing sublocalization of AIM2 to 1q22. The AIM2 consensus sequence shares strong similarity with known human interferon-inducible Expression of AIM2 genes IFI16 (P=3.9e-50), MNDA (P=3.0e-10), and the mouse I®204 gene (P=1.0e-46). The AIM2 cDNA As documented in Figure 4a, AIM2 demonstrated contains an ORF of 1032 bp corresponding to a dierential expression between the parental tumori- putative polypeptide of 344 amino acids. BLASTX genic UACC-903 melanoma cell line and its suppressed aligns the predicted AIM2 amino acid sequence with subline UACC-903(+6) (Trent et al., 1990; Ray et al., IFI16 (P=3.9e-50), MNDA (P=1.6e-52), and mouse 1996). Speci®cally, AIM2 was signi®cantly overex- I®204.(P=4.5e-47). Figure 2 shows the alignment of pressed following the introduction of human chromo- the AIM2 open reading frame with those of IFI16, some 6 and its expression coincided with reversion of MNDA and I®204. the tumorigenic phenotype (Ray et al., 1996). Because

Figure 1 The 1485 bp sequence of the AIM2 cDNA with the amino acid sequence of the predicted coding region indicated below the nucleotide sequence. The stop codon is indicated by an asterisk. Boxed nucleotides indicate a polyadenylation signal. The tyrosine kinase phosphorylation site is underlined Cloning of a novel interferon inducible gene KL DeYoung et al 455 of the dierential expression pattern in UACC- than in other tissues, and re-probing the same blots 903(+6), we examined AIM2 expression in normal with b-actin con®rmed this pattern of expression. melanocyte RNA by slot blot and, failed to recognize detectable levels in normal melanocytes (results not Induction of AIM2 in HL60, UACC-903 and UACC- shown). 903(+6) cells Preliminary information on the tissue distribution of AIM2 expression was obtained by using the AIM2 To examine the eect of IFN-g on AIM2 mRNA cDNA as a probe against a multiple tissue Northern expression; the interferon-inducible cell line HL60 was blot (Figure 4b). The expected 2 kb transcript was grown for varying periods of time in the presence of detected in total RNA from spleen, small intestine, and IFN-g (100 U/ml). Cells were harvested and RNA was peripheral blood leukocytes, and a slightly larger extracted from independent cultures following 0, 6, 12, transcript was detected in mRNA from testis. AIM2 24 or 36 h of IFN-g exposure. Northern blots were mRNA expression was signi®cantly higher in spleen prepared for hybridization with the AIM2, IFI16, MNDA, and cDNAs (Figure 5). AIM2 induction was of similar time course and magnitude to that of the related interferon inducible genes IFI16 and MNDA. In contrast to MNDA, induction of AIM2 by DMSO was not observed.

Figure 2 Amino acid alignment of AIM2, IFI16 (GenBank S75415S01), MNDA (GenBank HSU30245), and IFI204 (GenBank 31419) proteins. Conserved residues that are present in at least two of the four proteins are blocked. The letter X in Figure 3 Fluorescent in situ hybridization (FISH) to normal the consensus sequence indicates positions of nonidentity. The human metaphase chromosomes using an AIM2 genomic probe protein sequence alignment was created by the Lipman Pearson (see text). Inset, identical DAPI-banded chromosome 1 identi®ed program in DNASTAR software (version 1.58, DNASTAR, Inc.) by arrow following FISH. Results sublocalize AIM2 to 1q22

a b . 6) e UACC-903 UACC-903(+ D151JA Spleen Thymus Prostate Testis Ovary Sm. Intestin Colon Per .BI. Leuk Heart Brain Placenta Lung Liver Skel. Muscle Kidney Pancreas

2.0 kb —

2.4 kb — — β-actin

1.4 kb —

Figure 4 Expression of AIM2 in cell lines and human tissues. (a) Northern analysis of AIM2 expression in melanoma cell lines. The AIM2 probe detects the enhanced a 2 kb transcript in the parental (UACC-903), and two chromosome 6-suppressed melanoma cell lines (UACC-903(+6)-entire 6; D151JA-6q only). (b) Northern analysis of AIM2 expression in normal human tissues. Human b-actin was hybridized as a control for loading error Cloning of a novel interferon inducible gene KL DeYoung et al 456 domain which is present in MNDA and the interferon- γIFN inducible mouse gene D3 and which is present in duplicate in IFI16 and the I®200 genes. To date, all of 061224 36 D the genes from these families which share this — 28S conserved sequence are interferon-inducible, although no observable correlation exists between the number of IFI 16 — copies of the conserved domain and the type of interferon which regulates expression. For example, — 18S Briggs et al., (1994) note that while MNDA and D3 both contain a single copy of the conserved amino acid domain and are similar in patterns of expression and cell type-speci®c regulation, they are up-regulated by — 28S dierent forms of interferon (a and g, respectively). The expression pattern of AIM2 in normal human tissues, speci®cally in spleen and peripheral blood leukocytes, is consistent with data suggesting that — 18S AIM2 — genes in this family may be involved in the blood cell-speci®c response to interferons (Trapani et al., 1992; Tannenbaum et al., 1993; Briggs et al., 1994; — 28S Dawson et al., 1995). However, this gene is clearly expressed in non-lymphoid tissues, and the most striking novel feature of AIM2 expression is the ®nding of marked up-regulation of this gene in the MNDA — — 18S UACC-903(+6) cell line. This dierential expression was not observed with IFI16, leading us to speculate that these genes are regulated dierently in this model system. The role of AIM2 in suppression of the — 28S malignant phenotype is currently indeterminate. How- ever, studies are underway to assess the possibility that ETBR AIM2 may play a role in tumorigenic reversion. Finally, further studies appear warranted to provide — 18S insight into the biologic function of AIM2 as well as the signi®cance of human interferon-inducible genes in the model system. 12345 6 Figure 5 Northern blot analysis of RNA (10 mg) puri®ed from HL-60 cells incubated in medium containing 100 U/ml recombi- Materials and methods nant human gamma IFN for the number of hours indicated (lanes 1 ± 5) or with DMSO (lane 6). The blot was probed with IFI16 (a), AIM2 (b) and MNDA (c) cDNA probes, and the blot was Cell culture stripped between each separate hybridization. Equal amounts of HL60, UACC-903 and UACC-903(+6) cells (Trent et al., RNA were loaded into each lane as shown in d 1990) were cultured in RPMI 1640 media with 10% fetal bovine serum, 2 mML-glutamine, 100 units/ml penicillin G sodium, and 100 mg/ml streptomycin sulfate. UACC- Discussion 903(+6) harbors a psv2neo tag for selection of the introduced normal chromosome 6, requiring the addition In this study we have presented preliminary character- of 600 mg/ml G418. Normal human melanocytes (Clo- ization of a novel gene related to the interferon- netics, San Diego, California) were grown in media inducible IFI16 gene family which is dierentially supplied by the manufacturer. Melanocytes were grown expressed following the suppression of the tumorigenic for two weeks before harvesting for isolation of total phenotype in a malignant melanoma cell line. The RNA. For interferon induction studies, HL60 cells were nucleotide and predicted amino acid sequences incubated in medium containing gamma-interferon, 100 U/ ml (Amersham, Sydney, Australia) for the times indicated demonstrate homology not only to the human or with dimethylsulfoxide (DMSO), 1.25% (v/v) (Ajax interferon-inducible gene IFI16 (Trapani et al., 1992) Chemicals, Melbourne, Australia). but also to MNDA (Briggs et al., 1994), as well as to members of a family of interferon-inducible genes in mouse (Choubey et al., 1989). Like IFI16 and MNDA, Northern analyses AIM2 is inducible by IFN-g in HL60 cells. This data, Analysis of AIM2 expression in cultured cells was combined with cytogenetic mapping of AIM2 to performed using total RNA isolated either by guanidinium human chromosome band 1q22, supports the previous isothiocyanate/CsTFA centrifugation (Kingston et al., suggestion (Briggs et al., 1994) that a human gene 1991), Trill (Gibe BRA, Gaithersburg, MD), or RNAzol family, closely related to the mouse I®200 family, exists extraction (Biotecx Laboratories, Houston, TX) according to the manufacturers' recommendations. 10 mg(HL60)or within a linkage group that is conserved between 20 mg (melanoma cells) total RNA were electrophoresed human band region 1q21 ± 32 and distal mouse under denaturing conditions and transferred to Hybond chromosome 1 (Seldin et al., 1988a,b; Kingsmore et N+ or Nytran membranes (Amersham Life Science, al., 1989; Peters et al., 1989; Briggs et al., 1994). AIM2 Arlington Heights, IL; Schleicher and Schuell, Dassel, contains one copy of a 200 amino acid conserved Germany). Probes were generated by labeling cDNAs for Cloning of a novel interferon inducible gene KL DeYoung et al 457 IFI16, AIM2 or MNDA with [32P]. Hybridizations were (cytogenetic band and Flpter measurements) were obtained conducted at 428C overnight in hybridization solution for each hybridization signal in 20 metaphase chromo- containing 56 Denhardt's, 0.75 M,0.075MNa citrate, 1% somes using Band Map 2.2 (Burde et al., 1996) in NIH (w/v) SDS, 5% (w/v) dextran sulphate and 200 mg/ml Image. herring sperm DNA. Blots were washed twice in 26 SSC/ 0.1% SDS at 428C, twice in 0.26 SSC/0.1% SDS at 428C, Isolation and cDNA sequencing and twice in 0.16 SSC/0.1% SDS at 688C for 15 min per wash followed by exposure to Kodak K-Omat AR ®lm at The GENETRAPPER cDNA Positive Selection System 7808C. For IFN-g induction experiments, blots were from Gibco-BRL (Life Technologies, Gaithersburg, MD) washed to a strigency of 0.016SSC. Analysis of expres- was utilized with DNA from a spleen cDNA library. sion in normal human tissues was performed on commer- Single-stranded DNA was generated with Gene II protein cially prepared blots (Clontech, Palo Alto, CA) using the and Exonuclease III and subsequently hybridized to a manufacturer's recommended protocol. A b-actin probe biotin-tailed oligonucleotide probe. Target cDNA was wasusedasaloadingcontrol. captured using streptavidin-coated paramagnetic beads. Single-stranded target DNA was then primed and repaired to make a double-stranded DNA molecule. This repaired PCR mapping and P1 isolation DNA was used to transform E. coli by electroporation, and Preliminary chromosomal assignment was performed using candidate clones were isolated. Using vector primers and sequence-speci®c oligonucleotide primers against genomic cDNA-speci®c oligonucleotide primers (Genosys Bio- DNAs from the NIGMS Human/Rodent Somatic Cell technologies, Inc., The Woodlands, TX, and The Midland Hybrid Mapping Panel #2 (Coriell Institute for Medical Certi®ed Reagent Co., Midland, TX), cDNA subclones Research, Camden, NJ). A P1 phage library was screened were sequenced to establish a consensus for the AIM2 by PCR using primers 56B-11F4: 5'-GGG AAG CAG coding region. All clones were sequenced from both DNA GAG ATG TTT CAT G-3' and 56B-11B3: 5'-GGG GGA strands. Sequence analysis, including contig assembly, CAT TAA CCT TTT GTC AG-3', as described previously sequence alignments, and consensus formation, was (Ray et al., 1996). accomplished using DNA STAR software (version 1.58, DNASTAR Inc., Madison, WI). The AIM2 consensus was compared to previously cloned sequences using BLASTN Fluorescent in situ Hybridization (FISH) and BLASTX (Altschul et al., 1990). A P1 clone positively identi®ed by PCR using sequence- speci®c AIM2 primers was labeled with Spectrum-orange dUTP (Vysis, Napersville, IL) by nick-translation and used as a FISH probe for hybridization to metaphase Acknowledgements chromosomes from normal peripheral blood lymphocytes The authors acknowledge the assistance of Darryl Leja in as previously described (Pinkel et al., 1986). Map positions preparation of graphic illustrations.

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