Candidate tumor suppressor RIZ is frequently involved in colorectal carcinogenesis

Robert B. Chadwick*, Guang-Liang Jiang†, Garth A. Bennington*, Bo Yuan*, Cheryl K. Johnson*, Michael W. Stevens*, Theodore H. Niemann*, Paivi Peltomaki*, Shi Huang†, and Albert de la Chapelle*‡

*Division of Human Cancer Genetics and Department of Pathology, , Comprehensive Cancer Center, Columbus, OH 43210; and †Program in Oncogenes and Tumor Suppressor Genes, The Burnham Institute, La Jolla, CA 92037

Contributed by Albert de la Chapelle, December 28, 1999 The distal portion of chromosome 1p is one of the most commonly poly(A) tracts. RIZ encodes a retinoblastoma-interacting zinc affected regions in human cancer. In this study of hereditary and finger protein, and the RIZ1 isoform (but not RIZ2, an sporadic , a region of frequent deletion was alternative product of the same gene) is commonly lost or identified at 32.2 centimorgans from 1ptel. Deletion breakpoints expressed at reduced levels in cancer cells. Forced expression ͞ ͞ clustered in the vicinity of or inside the gene RIZ, which encodes a of RIZ1 causes cell-cycle arrest at G2 M and or apoptosis and retinoblastoma protein-interacting zinc finger protein. Sequence suppresses tumorigenicity in nude mice (10–12). Our obser- analysis revealed frequent frameshift mutations of the RIZ gene. vation of a high rate of involvement of RIZ through frameshift The mutations consisted of 1- or 2-bp deletions of a coding (A)8 or mutations and genomic loss suggests a role in colorectal (A)9 tract and were confined to microsatellite-unstable colorectal tumorigenesis. tumors, being present in 9 of 24 (37.5%) primary tumors and in 6 of 11 (54.5%) cell lines; in 2 cell lines the mutation was homozy- Materials and Methods gous͞hemizygous. The mutations apparently were selected Tissue Samples and Cell Lines. Among a total of 22 MSI(Ϫ) tumors clonally in tumorigenesis, because similar poly(A) tracts in other studied, 8 were selected because they previously had been found genes were not affected. Two alternative products of the gene to display a CIN phenotype, including LOH at two closely linked exist, RIZ1, which contains a PR (PRDI-BF1-RIZ1) domain implicated markers, D1S228 [32.4 centimorgans (cM)] and D1S507 (36.2 in tumor suppressor function, and RIZ2, which is lacking this motif. cM) (13). In 14 tumors, the LOH status at 1p was unknown. In Furthermore, the C-terminal region, which contains the poly(A) addition, we studied 24 MSI(ϩ) tumors from hereditary non- tracts, includes a PR-binding motif, possibly mediating interactions polyposis colorectal cancer (HNPCC) patients (14, 15). More- with other proteins or with RIZ itself (oligomerization). Four of over, 11 MSI(ϩ) and 3 MSI(Ϫ) cell lines were purchased from eleven microsatellite-unstable colorectal cancer cell lines, three of the American Type Culture Collection. The MSI(ϩ) lines were which had frameshifts, showed reduced or absent mRNA expres- DLD1, LS411N, SW48, HCT116, LoVo, HCT15, and LS174T sion of RIZ1. In a cell line that is homozygous͞hemizygous for the (colorectal cancer); MDAH2774 and SK-OV3 (ovarian cancer); typical frameshift mutation, immunoblotting showed truncated AN3CA (); and DU145 (prostate cancer). RIZ protein, whereas adenovirus-mediated RIZ1 expression caused The MSI(Ϫ) lines were MDAMB231, MDAMB435S, and G2͞M arrest and apoptosis. We propose that RIZ is a target of the SKBR3 (breast cancer), which had been previously characterized observed 1p alterations, with impairment of the PR domain- for RIZ expression (10). mediated function through either frameshift mutation or genomic deletion. LOH Analysis. Primary normal͞tumor pairs were investigated by using fluorescently labeled microsatellites. Primer sequences were obtained from the Genome Database (http:͞͞gdbwww.g- poradic cancer arises as a result of a series of somatic db.org͞). Amplifications of each microsatellite were done in mutations and epigenetic changes that silence tumor sup- S 15-␮l volumes with 10 ng of each respective genomic DNA, 8 pressor genes or activate oncogenes (1). As such, the APC-RAS- pmol of each primer (5Ј primer, fluorescently labeled), 100 ␮M P53 pathway in colorectal cancer is well established (2), but many each dNTP, 0.6 unit of AmpliTaq Gold DNA Polymerase (PE of its components are not fully understood (3). Recently, two Biosystems, Foster City, CA), 10 mM Tris⅐HCl (pH 8.3), 50 mM pathways, CIN, a chromosomal instability pathway, and MIN, a KCl, and 2 mM MgCl . PCR products were loaded onto a 377XL microsatellite instability pathway, have been proposed. Loss of 2 sequencer (PE Biosystems). Allele size and fluorescent intensity heterozygosity (LOH) is a hallmark of the CIN pathway, whereas were determined by GENESCAN and GENOTYPER software (PE microsatellite instability (MSI) characterizes the MIN pathway Biosystems). LOH was analyzed by determining the fluorescent (4). In the MIN pathway, mutations or epigenetic silencing of intensity of each allele and calculating the ratio (13). A sample one of the DNA mismatch repair genes lead to widespread was scored as showing LOH if an allelic ratio of Ͻ0.67 or Ͼ1.5 accumulation of mutations, followed by clonal selection (5). The ϩ was obtained. MSI( ) phenotype is easily distinguishable by allelic changes of Because of a high degree of MSI observed in the HNPCC repetitive regions throughout the genome (6). tumor DNAs, single-nucleotide polymorphisms (SNPs) were Some genes contain repetitive regions in their coding se- quences that are often targets of MSI. Insertion or deletion in these repetitive regions leads to frameshift and protein trun- Abbreviations: MSI, microsatellite-unstable; PR, PRDI-BF1-RIZ1; LOH, loss of heterozygosity; cation. In colorectal cancer, the TGFBRII gene (7) and BAX HNPCC, hereditary nonpolyposis colorectal cancer; SNP, single-nucleotide polymorphism; gene (8) are examples of genes mutated in this way and whose cM, centimorgan. loss of function is believed to contribute to tumorigenesis. ‡To whom reprint requests should be addressed at: Human Cancer Genetics Program, Ohio Other examples include the DNA mismatch repair genes State University, 420 West 12th Avenue, 6th Floor, Columbus, OH 43210. E-mail: de- [email protected]. MSH6 and MSH3, and IGFR2 (9). In this paper, we describe The publication costs of this article were defrayed in part by page charge payment. This how a high-density LOH analysis led to the identification of a article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. frequently deleted region in 1p36 comprising the RIZ gene. In §1734 solely to indicate this fact. ϩ MSI( ) tumors that did not show LOH, RIZ was found to be Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073͞pnas.040579497. affected by frequent frameshift mutations of one of two coding Article and publication date are at www.pnas.org͞cgi͞doi͞10.1073͞pnas.040579497

2662–2667 ͉ PNAS ͉ March 14, 2000 ͉ vol. 97 ͉ no. 6 Downloaded by guest on September 28, 2021 Downloaded by guest on September 28, 2021 Chadwick tal. et PNAS ͉ ac 4 2000 14, March ͉ o.97 vol. ͉ o 6 no. ͉ Fig. 1. Deletion mapping of chromosome 1p in HNPCC and sporadic colorectal cancers. Markers highlighted in red indicate LOH (allelic ratios of Ͻ0.67 or Ͼ1.5). Yellow highlighted 2663 markers show MSI. Blue highlighted markers are informative and show no LOH, and markers that are not highlighted are not informative. ⌬, Frameshift mutation in poly(A) repeat.

GENETICS also used to determine LOH in the subset of 24 HNPCC normal͞tumor DNA pairs. Primer sequences were obtained from the Human SNP database (http:͞͞www-genome.wi.mit- .edu͞SNP͞human͞index.html). SNPs were amplified in 25-␮l volumes with 100 nmol of each of the respective PCR primers, 25 ng of genomic DNA, 100 ␮M each dNTP, 1.0 unit of AmpliTaq Gold DNA Polymerase (Perkin–Elmer), 10 mM ⅐ Tris HCl (pH 8.3), 50 mM KCl, and 2 mM MgCl2. PCR products were purified by using exonuclease 1 and shrimp alkaline phosphatase (Amersham Life Sciences) and directly sequenced in one direction with one of the amplification primers and the BigDye Terminator chemistry (PE Biosys- tems). Samples that failed or sequenced poorly were rese- quenced in the other direction with the other amplification primer. LOH determination was done by a method similar to the microsatellite analysis.

Mutation Analysis. Candidate genes were screened for mutations by direct sequencing of genomic PCR products. To facilitate direct sequencing of PCR products, all primers were tailed with M13-forward (TGTAAAACGACGGCCAGT) and M13- reverse (CAGGAAACAGCTATGACC) sequences (primer se- quence is available from authors on request). PCRs were done ␮ in 25- l volumes with 100 nmol of each of the respective PCR Fig. 2. Example of LOH with SNP WIAF-481. A high number (8͞15 or 53.3%) primers, 25 ng of genomic DNA, 100 ␮M each dNTP, 1.0 unit ⅐ of informative HNPCC tumors display LOH with this SNP. This marker maps to of Taq Gold DNA polymerase (Perkin–Elmer), 10 mM Tris HCl 32.2 cM on the Human SNP database (http:͞͞www-genome.wi.mit.edu͞SNP͞ (pH 8.3), 50 mM KCl, and 2 mM MgCl2. PCR fragments were human͞index.html). purified by using the Exonuclease I͞Shrimp Alkaline Phospha- tase PCR Product Presequencing Kit (United States Biochem- ical). After purification according to the manufacturer’s markers closest to 1ptel were not informative, a region of protocol, 2 ␮l of the PCR products were sequenced by using common deletion was identified by the marker WIAF-481 at 32.2 the BigDye Terminator AmpliTaq FS Cycle Sequencing Kit cM, which showed LOH in 8 of 15 (53%) informative HNPCC (PE Biosystems). tumors (Fig. 1). The allelic imbalance values for SNPs were highly reproducible (Fig. 2), indicating the validity of these Expression Analysis. RNA for expression analysis was isolated by markers in the deletion mapping of MSI(ϩ) tumors in particular. using the RNAeasy Mini Kit (Qiagen, Chatsworth, CA). Reverse In comparison, a much lower rate of LOH was observed for transcription of isolated RNA was done by using Superscript RT nearby markers such as D1S450 (1͞11) at 22.9 cM and D1S228 (Life Technologies, Rockville, MD), and cDNA amplification was (1͞8) at 32.4 cM. done by using the GeneAmp Gold RNA PCR kit (PE Biosystems). Primers used for determination of RIZ1- and RIZ2-specific PCRs Identification of RIZ as a Candidate Gene for 1p Alterations. Func- were as described (10). Glyceraldehyde-3-phosphate dehydroge- tional candidate genes in the region of 32.2 cM from 1ptel were nase (GAPDH)-specific primers were used as a control (16). examined and screened for mutations. The RIZ gene (retino- blastoma protein-binding zinc finger protein) maps to 32.2 cM on Immunoprecipitation and Immunoblot Analysis. Immunoprecipita- GeneMap 99 (http:͞͞www.ncbi.nlm.nih.gov͞genemap99͞). RIZ tion and immunoblot analysis was performed according to is within 370 kb of D1S228, as inferred from yeast artificial previously described procedures (17). Cell extracts were immu- chromosome analysis (16). RIZ liesin3Ј to 5Ј orientation from noprecipitated with RIZ antiserum 1637 against the N terminus the telomere of chromosome 1p. In HNPCC tumors, there was of RIZ2 or preimmune serum. Immunoprecipitated products a gradual decline of the LOH rate from WIAF-481 to RIZ 3Ј to were resolved on a 5% SDS gel followed by immunoblot analysis RIZ 5Ј. Three intragenic polymorphisms of RIZ were analyzed using mouse antiserum KGSE against the N terminus of RIZ2. for LOH, including a codon Pro-704 deletion at exon 8, an intron 4 CA repeat (18), and a SNP flanking exon 4 (GAT to GAC 18 Analysis of Cells Expressing Ectopically Introduced RIZ1. This analysis bases 3Ј of the coding exon 4 sequence). The LOH rates for these was performed as described previously (10). Briefly, colon RIZ markers were 21% (3͞14), 9% (1͞11), and 0% (0͞8), cancer cells were seeded at 2 ϫ 105 cells in 6-cm dishes and respectively. Tumor 5393T showed LOH of exon 8 Pro-704 but infected with recombinant adenovirus at a multiplicity of infec- retention of the other intragenic markers, consistent with loca- tion of 100. At 48 hr postinfection, cells were processed for DNA tion of a deletion break point inside the RIZ gene. Analysis of histogram analysis. MSI(Ϫ) tumors that were not preselected for 1p LOH revealed LOH rates of 30% (4͞13), 23% (3͞13), and 60% (3͞5), respec- Results tively. Tumor 7T-OSU showed LOH of RIZP704 but retention Deletion Mapping of 1p. Most of the sporadic colorectal cancer of heterozygosity of RIZ intron 4 CA, again indicating a deletion tumors showed deletion of the entire 1ptel region up to Ϸ40 cM, breakpoint within RIZ. The location of RIZ in the vicinity of the where there was a return to heterozygosity in several tumors common LOH region and the observed deletion breakpoints (Fig. 1). The region of return to heterozygosity is telomeric to within RIZ suggest that RIZ is a candidate target of 1p36 several candidate tumor suppressor genes and oncogenes, in- alterations in both hereditary and sporadic colorectal cancers. cluding PAX7 at 46.2 cM, PLA2 at 46.2 cM, E2F2 at 52.4 cM, and Examination of the coding sequence of RIZ showed poten- Ϫ MYCL at 71 cM. In comparison with MSI( ) tumors, the area tially hypermutable tracts of (A)8 and (A)9 in exon 8 of the RIZ of shared deletion was more restricted in MSI(ϩ) tumors from gene. In 9 of 24 (37.5%) of the MSI(ϩ) HNPCC tumors, ϩ HNPCC patients (Fig. 1). Although the majority of the SNP frameshift mutations were found in either the (A)8 [one MSI( )

2664 ͉ www.pnas.org Chadwick et al. Downloaded by guest on September 28, 2021 Fig. 4. Expression analysis of the RIZ1 and RIZ2 isoforms in breast, colorectal, ovarian, and endometrial cancer cell lines. No RIZ1 transcript at all is detect- able in mutation-positive cell line LS411N (colon). Other mutation-positive cell lines show decreased expression of RIZ1, including LoVo (colon) and HCT116 (colon). Breast cancer cell lines MDAMB435S and SKBR3 are used as positive controls, because they have been previously reported to not express RIZ1 or to express it at a reduced level.

ϩ tumor; Fig. 3A] or the (A)9 [eight MSI( ) tumors; Fig. 3B] tract. Furthermore, 6 of 11 MSI(ϩ) cell lines (HCT116, LoVo, LS411N, LS174T, MDAH2774, and AN3CA) showed frameshift mutations in the (A)9 tract. In cell lines HCT116 and AN3CA, the mutation was homozygous͞hemizygous, resulting in biallelic involvement (Fig. 3C). None of the 23 tested MSI(Ϫ) sporadic colorectal cancers contained mutations in either of the polya- denosine tracts, indicating that these regions were mutational hotspots in MSI(ϩ) tumors only.

RIZ Frameshift Mutations Are Clonally Selected in Tumorigenesis. To investigate whether similar mononucleotide tracts in other genes are involved to a comparable degree, we studied the coding (A)8 tracts of the PMS2 and DNA polymerase ␣ genes. Frameshift mutations in these tracts were completely absent in the MSI(ϩ) GENETICS tumors (P ϭ 0.0005). MSI(ϩ) tumors were further screened for mutations in the (A)9 tracts of the RECQL, BLM, and KIAA0355 genes. One of 24 MSI(ϩ) tumors had 1-bp deletions in the RECQL and KIAA0355 genes (P ϭ 0.005). MSI(ϩ) tumors were screened further for mutations in the (A)9 tracts of the RECQL, BLM, and KIA0355 genes. Of 24 MSI(ϩ) tumors, 1 had 1-bp deletions in the BLM gene (P ϭ 0.005). Taken together, these results suggested that the RIZ frameshift mutations were spe- cifically selected during the clonal evolution of colorectal tumorigenesis.

Decreased RIZ1 Expression in Cell Lines. Previous reports have shown that RIZ is expressed in two alternative transcripts, RIZ1 and RIZ2 (10). RIZ1 is commonly lost, whereas RIZ2 is present uniformly in several 1p36-linked cancer types (10–12). To de- termine whether expression was affected in RIZ-mutated colo- rectal tumors, we studied expression by the reverse transcrip-

1-bp deletion in this repeat. MSI(ϩ) tumor sample 8201T (Lower) has a 2-bp deletion in this repeat. (C) Colon cancer cell line HCT116 is homozygous͞ Fig. 3. (A) Truncating frameshift mutation in the coding (A)8 repeat of exon hemizygous for a 1-bp deletion in the (A)9 repeat of exon 8 of RIZ, and 8 of the RIZ gene. MSI(ϩ) tumor sample H2AT (Lower) has a 1-bp deletion in endometrial cell line AN3CA has both 1- and 2-bp deletions in this repeat. this repeat. (B) Examples of truncating frameshift mutations in the coding (A)9 MSI(ϩ) colon cell lines LoVo, LS411N, and LS174T also have 1-bp deletions in repeat of exon 8 of the RIZ gene. MSI(ϩ) tumor sample 5381T (Center) has a this repeat, as does the ovarian cell line MDAH2774.

Chadwick et al. PNAS ͉ March 14, 2000 ͉ vol. 97 ͉ no. 6 ͉ 2665 Downloaded by guest on September 28, 2021 tion–PCR as described previously (10–12). Because of total overlap between the smaller RIZ2 and the larger RIZ1 tran- scripts, transcription cannot be measured for RIZ2 alone; hence, one reaction is specific for RIZ1, whereas the other measures RIZ1 ϩ RIZ2. Breast cancer cell line MDAMB435S was used as a control that expresses only the RIZ2 isoform, and MDAMB231 was used as a control cell line that expresses both RIZ1 and RIZ2 isoforms (10). MSI(ϩ) colorectal cancer cell lines (4 of 11) showed reduced or lost mRNA expression of RIZ1 in the presence of abundant RIZ1 ϩ RIZ2 transcript (Fig. 4), supporting previous observations that an imbalance in the amounts of RIZ1 and RIZ2 is associated significantly with malignancy (10–12). In three cell lines with altered RIZ1 expression, frameshift mutations in the polyadenosine tracts were present, including cell line HCT116, which is homozygous or hemizygous for this mutation. Of these cell lines, LS411N was found to express only the RIZ2 isoform, whereas LoVo showed reduced expression of RIZ1. Also, SW48 showed reduced or absent expression of RIZ1, but had no mutations in the aden- osine repeats of RIZ. The RIZ sequence is large (nearly 8 kb), and it is possible that there are other areas in the gene or in the promoter region that could be affected, resulting in altered RIZ expression in these cell lines. RNA isolated from frozen tissue from the mutation-positive Fig. 5. Immunoblot analysis of RIZ protein expression. Equal amounts of cell HNPCC tumors did not show clear expression changes of RIZ. extracts from DLD1 and HCT116 cells were immunoprecipitated by preim- This finding is likely caused by the contamination of normal mune serum (PI) or anti-RIZ serum (␣RIZ) 1637 as indicated at the top of each tissue in the isolated RNA tumor samples. Titration experiments lane. The immunoprecipitated products were resolved on a 5% SDS gel of cell-line RNA showed that even with a mixture of 90% followed by immunoblot analysis using mouse RIZ antiserum KGSE. RIZ2-expressing mRNA to 10% RIZ1- and RIZ2-expressing mRNA, the RIZ1 and RIZ2 isoform is PCR amplified (data not shown). content indicating apoptotic cell death (Fig. 6 and Table 1). However, few cells with sub-G1 DNA content were observed in ͞ Frameshift Mutation Leads to Expression of Truncated RIZ Proteins. DLD1. The results show that RIZ1 caused G2 M arrest and ͞ The frameshift caused by the deletion of one adenosine at the apoptosis in HCT116 cells, but only G2 M arrest in DLD1 cells. (A)9 tract (at nucleotide position 4700 of the RIZ1 coding sequence) is expected to cause the fusion of truncated RIZ1 and Discussion RIZ2 lacking the C-terminal 219 aa with a novel reading frame RIZ is a protein with two alternative forms of 280 kDa (RIZ1) of 76 aa. This would lead to the expression of mutant RIZ1 and and 250 kDa (RIZ2). Apart from the N-terminal PR (PRDI- RIZ2 that are 157 residues shorter than their wild-type coun- BF1-RIZ1) domain that is present in RIZ1 but not in RIZ2, the terparts. To confirm that such truncated RIZ1 and RIZ2 gene contains several other motifs of interest. These motifs proteins indeed were expressed from the mutant allele, immu- include an RB-binding motif that is related to that of the noprecipitation and immunoblot analysis was performed using adenovirus E1A oncoprotein, eight zinc finger motifs, and a the HCT116 cell line that is homozygous or hemizygous for the PR-binding motif in the C-terminal region (17, 20, 21). The PR frameshift mutation. We first confirmed the usual pattern of domain is necessary for the negative regulatory function of RIZ RIZ protein expression in DLD1 cells that expressed both RIZ1 (10, 17) and may be involved in chromatin-mediated transcrip- and RIZ2 mRNAs. In all tumor cell lines we have studied tional activation or repression (20, 21). The interaction of the PR previously, RIZ1 protein was at low levels and difficult to detect, domain with the C-terminal binding domain might be necessary whereas RIZ2 protein was at higher levels and readily detectable for homo- or hetero-oligomerization of RIZ, and for interactions (10, 17, 19). Similarly, in DLD1 cells, RIZ2 protein of 250 kDa with other proteins (21). Database comparison of the presently was detected, and RIZ1 protein was at low or undetectable levels identified hypermutable regions of the zinc finger region of the (Fig. 5). In contrast, full-length RIZ2 protein was not detected in the HCT116 cell line, but instead a shorter protein of Ϸ230 kDa was observed, consistent with truncation of RIZ2 protein by the frameshift mutation (Fig. 5). Although the experiment was not informative for RIZ1 protein, a truncated RIZ1 protein could be inferred from the results on RIZ2.

RIZ1 Causes G2͞M Arrest, Apoptosis, or Both in Colorectal Cancer Cell Lines. Adenovirus-mediated RIZ1 expression has been shown to ͞ cause G2 M arrest, apoptosis, or both in breast and liver cancer cell lines. We examined the effects of adenovirus-mediated RIZ1 expression on HCT116 and DLD1 colon cancer cell lines. Immunoblot analysis confirmed full-length RIZ1 protein ex- pression in both cell lines upon infection with AdRIZ1 at a multiplicity of infection of 100 (data not shown). The fraction of Fig. 6. Effects of adenovirus (Ad)-mediated RIZ1 expression on colorectal ͞ infected cells in G2 M increased significantly at 48 h after cancer cell lines HCT116 and DLD1. RIZ1 caused G2͞M arrest and apoptosis in AdRIZ1 infection in HCT116 and DLD1 cells. At 48 and 72 h HCT116 cells, but only G2͞M arrest in DLD1 cells, as shown by DNA content postinfection with AdRIZ1, HCT116 cells showed sub-G1 DNA analysis.

2666 ͉ www.pnas.org Chadwick et al. Downloaded by guest on September 28, 2021 Table 1. Effects of RIZ1 expression on colorectal cancer cell lines not have any full-length RIZ, whereas RIZ1 did not cause HCT116 and DLD1 apoptosis in DLD1 cells, which carry the wild-type RIZ gene. Cells, difference in % The results suggest that colon cancer cell lines with mutant RIZ may be more susceptible to RIZ1-induced apoptosis. ͞ Cell line Apoptosis G1 SG2 M How does this correlate with the lost or reduced transcription HCT116 ϩ25 Ϯ 2.0 Ϫ47 Ϯ 1.3 ϩ6.1 Ϯ 0.7 ϩ20 Ϯ 0.8 of RIZ1 in most cell lines with a frameshift mutation? We DLD1 ϩ9.5 Ϯ 1.2 Ϫ28 Ϯ 2.1 Ϫ2.2 Ϯ 0.5 ϩ26 Ϯ 1.7 speculate that, in some cases, hemi- or homozygous somatic mutation or loss of RIZ1-specific sequence may occur in the DNA histogram analysis was performed 72 h after adenovirus (Ad) RIZ1 or course of clonal evolution. We show that in several tumors with AdNull virus infection. The values shown represent the difference in percent- LOH in the region, the return to heterozygosity occurred in the age of cell populations between AdRIZ1- and AdNull-infected cells. The vicinity of or inside of RIZ. Presently available polymorphic ϩ increase caused by AdRIZ1 over AdNull is indicated by a plus sign ( ), and a markers are not informative enough to provide a full analysis of decrease is indicated by a minus sign (Ϫ). The values represent the mean Ϯ SD of three experiments. every tumor. Moreover, LOH is difficult or impossible to assess in many cell lines, because the alleles that were present in the germ line are not known. We entertain the hypothesis that the RIZ gene shows that this region is highly conserved even among function of RIZ may be impaired by somatic events in at least ϩ such evolutionarily divergent species as Saccharomyces cerevisiae two different ways. In MSI( ) tumors (MIN pathway), frame- Ј and Drosophila melanogaster. The (A)8 tract in exon 8 of the RIZ shift mutations in the 3 end of the gene interfere with the gene is immediately 5Ј of the most C-terminal zinc finger interaction between the C terminus of the protein and its Ϫ domain, and frameshift mutation in the (A)8 tract predicts N-terminal PR domain. In MSI( ) tumors (CIN pathway), termination of translation such that this zinc finger would be mutations or deletions of the PR domain of RIZ1 may have truncated. The (A)9 tract is 30 nt (10 aa) past this same zinc similar effects. In this series of tumors, RIZ was affected by either finger domain. Frameshift mutations in either tract are predicted LOH or frameshift mutation, but not both, suggesting that LOH to lead to the loss of the C-terminal domain of the RIZ protein and frameshift mutations had similar, alternative functions in that is involved in PR binding. Thus, it is likely that these RIZ-associated tumorigenesis. frameshifts would be highly deleterious to the function of the Previous studies have established that RIZ1 is often under- RIZ protein. expressed, whereas RIZ2 is present or overexpressed in cancer Several criteria have been proposed to determine whether an cells (10–12), but the exact genetic mechanisms underlying these affected gene is a true target of inactivation involved in tumor- changes is unknown. Our data suggest more than one mecha- igenesis (22). These criteria include a high frequency of inacti- nism. The fact that RIZ is altered in many cancers, and the fact vation, biallelic inactivation, involvement of the candidate MSI that it resides in a chromosomal region that is frequently lost in target gene in a bona fide growth-suppressor pathway, inactiva- many tumors, calls for closer scrutiny of RIZ in many cancers. tion of the same growth-suppression pathway in MSI-negative tumors through inactivation of the same gene or genes within the We thank for critical reading of the manuscript. We also same pathway, and functional suppressor studies of in vitro or in thank Lauri Aaltonen for help and Julie Meek, Samuel Richards, vivo models. RIZ shows a high degree of frameshift mutations Marilotta Turunen, and Xiao-Hong Yang for technical assistance. Our that, in some instances, are biallelic and followed by reduced work was supported by National Institutes of Health (NIH) Grant P30CA16058 to Ohio State University, Comprehensive Cancer Center; expression. RIZ is potentially involved in the Rb growth- ͞ NIH Grant CA67941 and European Commission Grant suppressor pathway, and RIZ1 expression causes G2 M cell- ͞ BMH4CT960772 to A.d.l.C.; NIH Grant CA82282 to P.P.; and NIH cycle arrest and or apoptosis and suppresses tumorigenicity in Grant R01-CA76146, Tobacco Related Disease Research Program nude mice (10–12). Our data show that RIZ1 expression caused Grant TRDRP-7RT0026, and Cancer Research Program of California ͞ G2 M arrest and apoptosis in the HCT116 cell line, which does Grant CCRP-1II0023 to S.H. GENETICS

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