Oncogene (2001) 20, 980 ± 988 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc

Cloning and characterization of a senescence inducing and class II tumor suppressor gene in ovarian carcinoma at chromosome region 6q27

Francesco Acquati1,8, Cristina Morelli2,8, Ra€aella Cinquetti1, Marco Giorgio Bianchi1, Davide Porrini1, Liliana Varesco3, Viviana Gismondi3, Romina Rocchetti4, Simona Talevi4, Laura Possati4, Chiara Magnanini2, Maria G Tibiletti5, Barbara Bernasconi5, Maria G Daidone6, Viji Shridhar7, David I Smith7, Massimo Negrini2, Giuseppe Barbanti-Brodano2 and Roberto Taramelli*,1

1Dipartimento di Biologia Strutturale e Funzionale, Universita' dell'Insubria, Varese, Italy; 2Dipartimento di Medicina Sperimentale e Diagnostica, Sezione di Microbiologia, UniversitaÁ di Ferrara, I-44100 Ferrara, Italy; 3Istituto Nazionale per la Ricerca sul Cancro Genova, Italy; 4Istituto di Scienze Biomediche, UniversitaÁ di Ancona, I-60131 Ancona, Italy; 5Laboratorio di Anatomia Patologica, Ospedale di Circolo, Varese, Italy; 6Dipartimento Oncologia Sperimentale, Istituto Nazionale Tumori, Milano, Italy; 7Division of Experimental Pathology, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN 55905, USA

Cytogenetic, molecular and functional analysis has Introduction shown that chromosome region 6q27 harbors a senes- cence inducing gene and a tumor suppressor gene Abnormalities of the long arm of chromosome 6 are involved in several solid and hematologic malignancies. associated with several solid neoplasms including We have cloned at 6q27 and characterized the carcinomas of the ovary (Saito et al., 1992; Foulkes RNASE6PL gene which belongs to a family of et al., 1993; Cooke et al., 1996; Orphanos et al., 1995; cytoplasmic RNases highly conserved from plants, to Tibiletti et al., 1996), breast (Develee et al., 1991; man. Analysis of 55 primary ovarian tumors and several Theile et al., 1996; Chappel et al., 1997), uterus ovarian tumor cell lines indicated that the RNASE6PL (Tibiletti et al., 1997), stomach (Queimado et al., gene is not mutated in tumor tissues, but its expression is 1995), liver (DeSouza et al., 1995), colon and rectum signi®cantly reduced in 30% of primary ovarian tumors (Honchel et al., 1996), kidney (Morita et al., 1991), and in 75% of ovarian tumor cell lines. The promoter parathyroid gland (Tahara et al., 1996), melanoma region of the gene was una€ected in tumors cell lines. (Millikin et al., 1991) and hematological malignancies Transfection of RNASE6PL cDNA into HEY4 and such as non-Hodgkin B-cell lymphoma (Gaidano et al., SG10G ovarian tumor cell lines suppressed tumorigeni- 1992) and acute lymphoblastic leukemia (Hayashi et city in nude mice. When tumors were induced by al., 1990). Among the solid tumors, ovarian cancers RNASE6PL-transfected cells, they completely lacked have been the focus of intense studies, because very expression of RNASE6PL cDNA. Tumorigenicity was little is known about the genetic pathways underlying suppressed also in RNASE6PL-transfected pRPcT1/ ovarian carcinogenesis. Moreover, ovarian tumors are H6cl2T cells, derived from a human/mouse monochro- still associated with a rather high mortality rate, mostly mosomic hybrid carrying a human chromosome 6 deleted due to delay in diagnosis which usually is performed at at 6q27. Moreover, 63.6% of HEY4 clones and 42.8% the late stages of the disease. The elucidation of the of the clones of XP12ROSV, a Xeroderma pigmentosum genetic events leading to inactivation of tumor SV40-immortalized cell line, transfected with RNA- suppressor genes or activation of oncogenes in ovarian SE6PL cDNA, developed a marked senescence process tumors is therefore greatly needed. during in vitro growth. We therefore propose that By employing the loss of heterozygosity (LOH) assay RNASE6PL may be a candidate for the 6q27 senescence with markers localized along the entire long arm of inducing and class II tumor suppressor gene in ovarian chromosome 6, several consensus regions of loss have cancer. Oncogene (2001) 20, 980±988. been delineated. From centromere to telomere the order of these regions is 6q21 ± 23.3 (Orphanos et al., Keywords: ovarian carcinoma; senescence; chromosome 1995; Shridhar et al., 1999), 6q25.1 ± 25.2 (Colitti et al., 6q27; Class II tumor suppressor gene; ribonuclease 1998) and 6q26 ± 27 (Saito et al., 1992; Foulkes et al., 1993; Cooke et al., 1996; Orphanos et al., 1995; Tibiletti et al., 1996). The latter region has been deeply investigated and ®ne mapping has disclosed a genomic *Correspondence: R Taramelli interval ¯anked by markers D6S193 and D6S149 as the 8 Francesco Acquati and Cristina Morelli contributed equally to this shortest region lost in ovarian tumors (Cooke et al., work. Received 24 October 2000; revised 12 December 2000; accepted 14 1996; Saito et al., 1996). Deletions at 6q27 were December 2000 detected in 18 out of 20 benign ovarian tumors Tumor suppressor and senescence-inducing gene at 6q27 F Acquati et al 981 (Tibiletti et al., 1998), indicating that alterations in one allele or alleles (most of the tumors were aneuploid for or more genes mapping in this region are one of the chromosome 6) are expected to be altered by earliest events in ovarian carcinogenesis. inactivating mutations or small deletions. Using exon Furthermore, the survey of more than 12 studies, 1, 2, 7, 8 and 9 primer pairs, several abnormal SSCP reporting allele losses in ovarian carcinomas, allowed bands were observed. Sequence analysis revealed the the identi®cation of an even shorter region, de®ned by presence of a missense common variant in exon 9 (a C markers D6S193 and D6S297 which is included in the to T at nucleotide position 708 with respect to the greater D6S193 ± D6S149 interval (Cooke et al., 1996). ATG replacing Arg with Trip). In addition, three silent The analysis of the critical D6S193 ± D6S149 interval base substitutions in exon 1, 8 and 9 and two common resulted in the construction of a long range physical single-base changes in intron 1 and 6 were detected. It map and a YAC contig of the region (Tibiletti et al., was possible to con®rm the germline origin only of 1998; Hauptschein et al., 1998). Physical measurements exon 9 and intron 1 modi®cations. Most likely these indicate that the distance between D6S193 and D6S149 sequence variants are common polymorphisms be- is approximately 900 Kb, whereas the smaller cause, at least in some cases, the same alterations were D6S193 ± D6S297 interval spans about 100 Kb (Tibi- found to be present in the corresponding germ line. letti et al., 1998; Hauptschein et al., 1998). Within the Also, the 5' and 3' regions of the gene, investigated by latter genomic region we have recently mapped, cloned sequence analysis for the extension of 2 Kb, were and characterized a cDNA (RNASE6PL) (Trubia et found to be intact. On the whole, this analysis indicates al., 1997; Acquati et al., 2000) coding for a protein that the RNASE6PL gene is not a€ected by mutations with a great similarity to a class of highly conserved in primary ovarian cancers. Furthermore, the HEY4 cytoplasmic RNases present in plants (Anderson et al., and OVCAR3 ovarian cancer cell lines were also 1986; Clark et al., 1990), animal viruses (Schneider et investigated, but as in the primary tumors no al., 1993), bacteria (Meador and Kennell, 1990), fungi mutations were found. (McClure et al., 1989), drosophila (Hime et al., 1995) and mouse (Trubia et al., 1997). Interestingly, although Expression of RNASE6PL in normal tissues, primary these enzymes from di€erent organisms ful®ll di€erent ovarian tumors and ovarian tumor cell lines biological functions, a common feature of their activity is the control of cell growth (McClure et al., 1990). Since the RNASE6PL gene does not seem to be This e€ect is particularly challenging due to the mutated in primary ovarian tumors and tumor cell chromosomal location of the human RNASE6PL gene lines, we proceeded to study its expression in normal at 6q27. Indeed, RNASE6PL may be a candidate and neoplastic tissues. The 1.4 Kb message of the tumor suppressor gene whose loss could contribute to RNASE6PL gene is rather ubiquitous being present, the development or progression of ovarian cancers. We although at variable levels, in all tissues that were have assessed such potential role for RNASE6PL and examined (Figure 1a). When fourteen ovarian cell lines we present here molecular and functional data were studied and compared with normal ovarian tissue, supporting an involvement of this gene in senescence several changes in RNASE6PL expression were and in ovarian tumorigenesis. detected. Figure 1b shows a representative example of eight cell lines where a reduced signal of RNASE6PL mRNA was detected in ®ve samples (HEY4, OV1225, OV166, SW626, OVRS1000), while in one cell line Results (SG10G) the expression of the gene was totally abolished and in two cell lines (SKOV3 and OVCR3) Genomic organization of the RNASE6PL gene was greatly enhanced. A series of 55 primary ovarian The structure of the RNASE6PL gene was determined tumors were also analysed and decreased expression of by YAC subcloning (Tibiletti et al., 1998, Hauptschein et RNASE6PL mRNA, below 25% of the normal al., 1998) and cosmid assembling. This gene, which is ovarian tissue, was detected in 15 (30%) of them. As present in a single copy in the genome, spans approxi- shown in Figure 1c, some samples displayed easily mately 27 Kb of genomic DNA and is split into nine detectable levels of RNASE6PL mRNA, whereas in exons and eight introns (the accession number for the other samples the signal was barely detectable. genomic sequence of the gene is AL159163). Transfection of tumor and immortal cell lines, induction of Mutation analysis of the RNASE6PL gene in primary senescence and suppression of tumorigenicity ovarian cancers In order to verify whether the RNASE6PL gene Fifty-®ve microdissected primary ovarian tumors, suppresses in vitro the transformed and immortal classi®ed from stage II to stage IV, were selected for phenotype, we transfected the ovarian cancer cell lines this analysis. These tumors were already known to HEY4 and SG10G as well as the SV40-immortalized contain 6q26-qter deletions in at least one of the cell line XP12ROSV with the pcDNA/Neo-RNS6 chromosome 6 homologues (Tibiletti et al., 1996; vector, containing the full-length human RNASE6PL Tibiletti and Taramelli, unpublished results). According cDNA under the control of the cytomegalovirus to the Knudson hypothesis, the remaining RNASE6PL immediate-early promoter. Suppression of tumorigeni-

Oncogene Tumor suppressor and senescence-inducing gene at 6q27 F Acquati et al 982 Table 1 shows a summary of the results of transfection of the RNASE6PL cDNA into pRPc, HEY4, SG10G and XP12ROSV cells. Some character- istics of the results were common among the transfected cell lines. In fact, the number of living clones that could be propagated in culture, compared to the number of clones picked up after selection, was constantly lower in cells transfected with the RNA- SE6PL cDNA inserted in sense orientation (S) than in cells transfected with the RNASE6PL cDNA inserted in antisense orientation (AS) or with the vector alone, suggesting that the RNASE6PL cDNA exerts an inhibitory e€ect on cell growth and cloning eciency. Moreover, all the clones transfected with the RNA- SE6PL cDNA-AS or with the vector alone were tumorigenic, whereas the pRPc and HEY4 cell clones transfected with the RNASE6PL cDNA-S were totally or partially suppressed in tumorigenicity (Tables 2 and 3). A greater tumorigenic activity was displayed by the clones of SG10G transfected with the RNASE6PL cDNA-S (Table 4). However, the tumors induced both by HEY4 and SG10G cell clones transfected with the RNASE6PL cDNA showed complete lack of expres- sion of the inserted cDNA, indicating that the exogenous RNASE6PL cDNA was no longer func- tional in the neoplastic tissue (Tables 3 and 4, Figure 2). Finally, while pRPc and SG10G cells showed a normal phenotype in vitro, 63.6% of HEY cell clones and 42.8% of XP12ROSV clones displayed a senescent phenotype. The analytical examination of the results of the Figure 1 Expression pattern of RNASE6PL in normal and single transfected cell lines indicates that pRPc normal 32 cancer cells. P-labeled RNASE6PL and GAPDH cDNA probes cells and pRPc cells transfected with the empty vector were used for Northern blot hybridizations of a commercial poly (A)+RNA ®lter representing several human tissues (a), a ®lter were fully tumorigenic, whereas pRPc cells transfected containing 25 mg per lane of total RNA from eight ovarian with the RNASE6PL cDNA-S produced tumors in cancer-derived cell lines and a normal ovarian tissue (b), and a three out of eight inoculated animals (Table 2). Of ®lter containing 20 mg per lane of total RNA from sixteen these three tumors, two completely regressed 30 days primary ovarian carcinomas and a normal ovarian tissue (c). Radioactive band intensity from each lane was measured by after inoculation and the third failed to grow in means of a PhosphorImage device and the RNASE6PL culture, indicating a limited viability of the tumor expression levels were calculated for each sample following cells. normalization with the signals obtained with the GAPDH control Transfection of HEY cells with the RNASE6PL probe cDNA-S yielded two classes of clones, since, out of eleven clones expressing the inserted cDNA, seven (63.6%) showed a senescent phenotype in vitro, while city was assessed in nude mice by inoculation of four (36.4%) had a normal phenotype. The senescent transfected HEY4 and SG10G cells as well as by phenotype gradually appeared and progressed during inoculation of pRPc cells cotransfected with the in vitro propagation of cell clones expressing RNA- plasmids pMT89, containing the RNASE6PL cDNA, SE6PL. Senescence was characterized by large and and pGK (see Materials and methods). These cell lines ¯attened cells, development of cytoplasmic vacuoles were selected for transfection for the following reasons: and nuclear shrinkage (Figure 3 a ± c). The process of the HEY4 cells show an expression of RNASE6PL senescence was documented by a positive beta- reduced to 10% of normal ovarian tissue (Figure 1b), galactosidase reaction (Figure 3 d ± g) and by a while the SG10G and XP12ROSV cells completely lack decreased incorporation of [3H]-thymidine in senescent expression of the RNASE6PL gene. The SG10G cell cells: at 72 h the mean value of thymidine incorpora- line was found to miss completely chromosome 6 as tion for three senescent clones was 245.6+89.8 c.p.m. evidenced by conventional cytogenetic and FISH as compared to 900.5+267.0 c.p.m. for three clones analyses (data not shown). The pRPc cells are mouse showing a normal in vitro growth phenotype (P50.05). cells derived from a tumor induced in nude mice by the When tumorigenicity was tested, four out of eight human/mouse monochromosomic hybrid pRPcT1/H6 inoculated cell clones, expressing RNASE6PL cDNA, and harbor a human chromosome 6 deleted at 6q27 were suppressed in tumorigenicity. Of the nine tumors (Gualandri et al., 1994). induced by the four tumorigenic clones, two completely

Oncogene Tumor suppressor and senescence-inducing gene at 6q27 F Acquati et al 983 Table 1 Summary of the results of transfection of the RNASE6PL cDNA into pRPc, HEY4, SG10G and XP12ROSV cells Cell line DNAa Clone (s) Pick (ed) Livingb DNAc RNAd In vitro phenotype Tumorigenicitye pRPc RNASE6PL-S 29 29 25 (86.2%) 3 (12.0%) 2 (66.7%) Normal 100% 2 PS pRPc PGK 4100 6 6 (100%) NA NA Normal 100% 1 T HEY4 RNASE6PL-S 78 78 47 (60.2%) 22 (46.8%) 11 (50.0%) Normal 36.4% 4 S; 3 PS; 1 T Senescent 63.6% HEY4 RNASE6PL-AS 4100 24 24 (100%) 8 (33.3%) ND Normal 100% 3 T HEY4 pcDNA3 4100 4 4 (100%) NA NA Normal 100% 3 T SG10G RNASE6PL-S 4100 68 49 (72.1%) 13 (26.5%) 6 (46.1%) Normal 100% 1 PS; 4 T SG10G RNASE6PL-AS 4100 24 22 (91.7%) 7 (31.8%) ND Normal 100% 2T SG10G pcDNA3 4100 4 4 (100%) NA NA Normal 100% IT XP12ROSV RNASE6PL-S 4100 97 10 (10.3%) 8 (80.0%) 7 (87.5%) Normal 57.2% ND Senescent 42.8% XP12ROSV RNASE6PL-AS 85 10 6 (60.0%) 5 (83.3%) ND Normal 100% ND XP12ROSV pcDNA3 4100 6 4 (66.7%) NA NA Normal 100% ND aS and AS refer to the RNASE6PL cDNA inserted into the expression vectors in sense or antisense orientation; bPercentage of living clones over picked clones; cPercentage of RNASE6PL DNA positive clones over living clones; dPercentage of RNASE6PL expressing clones over DNA positive clones; eNumber of inoculated cell clones which were tumorigenic or suppressed in tumorigenicity (T=tumorigenic; S=suppressed; PS=partially suppressed). ND, not done. NA, not applicable

Table 2 In vitro phenotype and tumorigenicity of pRPc cells after Table 3 In vitro phenotype and tumorigenicity of HEY4 cells after transfection of the RNASE6PL cDNA transfection of the RNASE6PL cDNA Clone DNAa RNAa Phenotype Tumorigenicityb Clone DNAa RNAa Phenotype Tumorigenicityb pRPc-S9 + + Normal 1/4 (19)c SA3c + + Senescent 2/3 (56) pRPc-S11 + + Normal 2/4 (26)d SA8d + ± Normal 3/3 (20) pRPc-S7 + ± Normal ND SB1 + + Normal 0/3 pRPc-pGKA3 NA NA Normal 4/4 (24.5) SB2 + + Normal 1/3 (48) pRPc NA NA Normal 4/4 (18.5) SB10 + + Senescent ND SB11 + + Senescent ND aDNA and RNA refer to RNASE6PL cDNA and RNA. bIn SC7 + + Senescent 0/2 parenthesis the mean value of the latency period for the appearance SC8 + + Senescent ND of tumors. cThis tumor completely regressed 30 days after SC10 + + Normal 0/3 development. dOne tumor regressed 30 days after development, the SD4 + + Senescent 2/3 (27)e other failed to grow in culture. ND, not done. NA, not applicable SD14 + + Normal 4/4 (24) SD17 + + Senescent 0/3 AS cl.2 + ND Normal 4/4 (45) AS cl.8 + ND Normal 4/4 (21) regressed (Table 3). Of the seven remaining tumors, AS cl.12 + ND Normal 3/3 (19) three, derived from clones SA3, SB2 and SD14 (Table pcDNA3A1 + NA Normal 4/4 (26) pcDNA3B1 + NA Normal 4/4 (22) 3), were tested by RT ± PCR and found to completely pcDNA3C1 + NA Normal 4/4 (17) lack insert expression (Figure 2a), thereby con®rming HEY4 NA NA Normal 4/4 (21) the tumor suppressing activity of the RNASE6PL cDNA. Three clones transfected by RNASE6PL-AS, aDNA and RNA refer to RNASE6PL cDNA and RNA. bIn parenthesis the mean value of the latency period for the appearance three clones transfected with the pcDNA3 vector alone of tumors. cS and AS refer to the RNASE6PL cDNA inserted into and one clone transfected by RNASE6PL-S, but not the vector pcDNA3 in sense or antisense orientation. dClone SA8, expressing the inserted cDNA, were tumorigenic (Table which does not express RNASE6PL cDNA, was used as a control in 3). Two clones, SB1 and SC10, showing a normal tumorigenicity assays. eThe two tumors completely regressed 30 and phenotype in vitro, were suppressed in tumorigenicity 41 days after development. The two tumors induced by clone SA3, the tumor induced by clone SB2 and two tumors induced by clone (Table 3), suggesting that senescence and suppression SD14 were assayed by RT ± PCR for the expression of RNASE6PL of tumorigenicity were induced through two di€erent cDNA with negative results (see Figure 2A). ND, not done. NA, not pathways by the RNASE6PL cDNA. Alternatively, the applicable two phenotypes may depend on a quantitative di€erence in the expression of the RNASE6PL cDNA. Indeed, dot blot analysis of clones with the senescent Transfection of SG10G cells with RNASE6PL and the normal phenotype showed that the senescent yielded six clones expressing the inserted cDNA (Table clones constantly exhibited a greater expression (twice 4). The in vitro phenotype of these clones was normal to three times) of the inserted cDNA, as compared to and no signs of senescence were detected. Five of the clones with a normal phenotype which are suppressed six expressing clones, tested for tumorigenicity, were in tumorigenicity (data not shown). This suggests that tumorigenic. However, when tumors, representative of senescence is induced only when the RNase cDNA is each tumorigenic clone, were analysed by Northern expressed in HEY4 cells over a certain threshold, while blot hybridization, they were all found to be negative suppression of tumorigenicity requires only a minimal for the expression of the 1.4 Kb RNASE6PL transcript expression of the insert. (Figure 2b). Two SG10G clones transfected with the

Oncogene Tumor suppressor and senescence-inducing gene at 6q27 F Acquati et al 984 Table 4 In vitro phenotype and tumorigenicity of SG10G cells after RNASE6PL-AS cDNA, one clone transfected with the transfection of the RNASE6PL cDNA vector alone and two clones transfected with RNA- Clone DNAa RNAa Phenotype Tumorigenicityb SE6PL-S, but not expressing the transfected cDNA, SA8 + + Normal 4/4 (18) were fully tumorigenic. SB13 + + Normal 3/3 (18) SB30 + + Normal ND SB31c + ± Normal 2/2 (5) CpG island methylation analysis of the RNASE6PL gene SD43 + + Normal 1/3 (33) in ovarian cell lines and primary carcinomas SD46 + + Normal 3/3 (22) SE49 + + Normal 2/2 910) Hypermethylation of normally unmethylated CpG SE53c ± ND Normal 2/2 (5) islands in the promoters and 5' ¯anking regions of genes, ASA3 + ND Normal 4/4 (12) down-regulated in cancers, is associated with loss or ASA10 + ND Normal 4/4 (10) reduction of (Jones and Laird, 1999; Baylin and Herman, pcDNA3D3 NA NA Normal 4/4 (10) SG10G NA NA Normal 4/4 (19) 2000). In order to carry out methylation studies, we determined the cap site, the proximal promoter region aDNA and RNA refer to RNASE6PL cDNA and RNA. bIn and more than 1 Kb of 5'-¯anking sequences of the parenthesis the mean value of the latency period for the appearance RNASE6PL gene. The region analysed spans the area of c of tumors. Clone SB31, which does not express RNASE6PL, and greatest CpG density immediately close to the transcrip- clone S53, which does not contain the insert, were innoculated as controls in tumorigenicity assays. Two tumors induced by clone SA8, tion start site of RNASE6PL. Normal ovarian tissue, the three tumors induced by clones SB13 and SD46, the tumor tumor cell lines and primary cancers were analysed by a induced by clone SD43 and one tumor induced by clone SE49 were methylation-speci®c PCR assay (Herman et al., 1996) assayed by Northern blot analysis for the expression of RNASE6PL which revealed that RNASE6PL is present both in the with negative results (see Figure 2b). ND, not done. NA, not applicable methylated and unmethylated form in the same sample. No di€erences were detected between normal and tumor samples. Moreover, the HEY4, SW626 and OV166 cell lines were treated with the demethylating agent 5-aza- cytidine to see whether RNASE6PL expression could be increased but no changes were observed. These data suggest that molecular mechanisms other than promoter methylation may control RNASE6PL expression in primary tumors and tumor cell lines.

Discussion

In this study we have investigated the possible role of RNASE6PL as a tumor suppressor gene in ovarian cancer. The gene was cloned at 6q27 (Trubia et al., 1997; Acquati et al., 2000), a chromosome region often deleted in ovarian tumors and in di€erent human neoplasms, such as melanoma, breast cancer, non- Hodgkin B-cell lymphoma and several others (Saito et al., 1993; Foulkes et al., 1993; Cooke et al., 1996; Orphanos et al., 1995; Tibiletti et al., 1996; Develee et al., 1991; Theile et al., 1996; Chappel et al., 1997; Queimado et al., 1995; DeSouza et al., 1995; Honchel et al., 1996; Morita et al., 1991; Tahara et al., 1996; Figure 2 Lack of human RNASE6PL expression in mouse Millikin et al., 1991; Gaidano et al., 1992; Hayashi et tumors. Total RNA was puri®ed from several tumor samples al., 1990). RNASE6PL belongs to a group of RNases obtained following s.c. injection in nude mice of HEY4 (a) and which are very conserved among species (Trubia et al., SG10G (b) clones which were previously selected in culture 1997; Anderson et al., 1986; Clark et al., 1990; following transfection of an expression vector containing human RNASE6PL cDNA. Total RNA from HEY4-derived tumors (a) Schneider et al., 1993; Meadoe and Kennell, 1990; was analysed for RNASE6PL expression by RT ± PCR, rather McClure et al., 1989; Hime et al., 1995). These enzymes than Northern analysis due to the presence of endogenous display di€erent functions. However, one of their main RNASE6PL transcripts in this cell line. Lane 1 : 1 kb DNA e€ects is regulation of cell growth (McClure et al., ladder standard; lanes 2 ± 4: tumor samples representative of HEY4 clones SD14, SA3 and SB2, respectively (see Table 4); lane 1990). In particular, a plant RNase, highly homologous 5: RNASE6PL expression vector used for transfection of cell to RNASE6PL, is responsible for the expression of lines; lane 6: negative control. The RNA samples from SG10G- gametophytic self-incompatibility in Nicotiana alata derived tumors (b) were analysed by Northern blot hybidization (McClure et al., 1990). This enzyme exerts its action by 32 with P-labelled RNASE6PL and GAPDH cDNA probes. The inhibiting the growth of pollen tubes through degrada- tumor analysed were derived from the following clones: lane 1: SA8; lanes 2 ± 4: SB13; lanes 5 ± 7: SD46; lane 8: SD43; lane 9: tion of pollen rRNA (McClure et al., 1990) with SE49; lane 10: placenta total RNA consequent inhibition of protein synthesis. It may be

Oncogene Tumor suppressor and senescence-inducing gene at 6q27 F Acquati et al 985

Figure 3 (a and b) Normal HEY4 cells. Magni®cation: a,256; b,506. c: HEY4 cell clone transfected with RNASE6PL cDNA and showing a senescent phenotype characeterized by ¯attened and enlarged cells with cytoplasmic vacuoles. Magni®cation: 506. Note that b and c have the same magni®cation. d±g: Four cell clones transfected with the RNASE6PL cDNA show a positive reaction for beta-galactosidase. Magni®cation: 506 conceivable that a similar mechanism could also be as local chromatin alterations or mutations in a gene(s) operative in the mammalian system, although at acting upstream of RNASE6PL. present we have no experimental evidence for this Furthermore, RNASE6PL-transfected ovarian tu- e€ect. On the other hand, it is worth noting that mor cell lines, inoculated into nude mice, were RNases like onconase (Ardelt et al., 1991) and BS- suppressed in tumorigenicity. All the induced tumors RNase (D'Alessio, 1993) are potent inhibitors of cell that were analysed resulted completely negative for the proliferation and the inhibitory e€ect is much more expression of the RNASE6PL RNA, con®rming the evident on actively growing cells, such as immortalized role of this gene in controlling tumor growth. or neoplastic cells (Schein 1997), lending support to the Due to its behavior in tumors, RNASE6PL may be potential role of these enzymes as tumor suppressors. de®ned as a class II tumor suppressor gene. Indeed, Indeed, onconase is currently being evaluated for the tumor suppressor genes are distinguished into class I treatment of mesothelioma in phase III clinical trials and class II. Class I tumor suppressor genes lose (Smith et al., 1999). function by deletion, rearrangement or mutation A mutation analysis on 55 ovarian tumors from (Haber and Harlow, 1997); class II tumor suppressor stage II to stage IV indicated the absence of mutations genes are structurally intact in their sequence, but are in the RNASE6PL coding region as well as in the 5' underexpressed or unexpressed, due to downregulation and 3' regulatory regions. Only a few polymorphisms or silencing in transcription or translation (Jones and were detected. However, analysis of the same ovarian Laird, 1999; Baylin and Herman, 2000). Loss of tumors by Northern blot hybridization showed absence expression may in turn depend on alterations a€ecting or reduction of RNASE6PL expression in 30% of the an upstream regulatory gene or on epigenetic mechan- samples as compared to normal ovarian tissue. isms altering the function of the class II gene promoter. Similarly, expression of the gene was reduced in 75% It is notable that all the genes which have convincingly of ovarian tumor cell lines. In order to shed light on been associated with tumor suppressing activity in the possible mechanisms responsible for such down- human ovarian neoplasms belong to class II. Indeed no regulation we have investigated whether the promoter inactivating mutations in primary tumors and cell lines of the RNASE6PL gene was methylated. Promoter were reported for GPC3 (Lin et al., 1999), NOEY2 (Yu methylation does not seem to account for the reduced et al., 1999), OVCA1 (Bruening et al., 1999), SPARC RNASE6PL expression as most of the tumors, cell (Mok et al., 1996) DOC-2 (Mok et al., 1998), and lines and normal ovary display both methylated and ZAC/LOT1 genes (Abdollahi et al., 1997; Bilanges et unmethylated alleles, without clear di€erences between al., 1999). The GPC3 gene promoter was found to be normal and tumor tissues. Most likely other mechan- methylated in several ovarian cancer cell lines (Lin et isms are acting in the downregulation of this gene such al., 1999), whereas the normal allele of the imprinted

Oncogene Tumor suppressor and senescence-inducing gene at 6q27 F Acquati et al 986 NOEY2 gene is always deleted in tumors showing analysis in this study. The HEY4 cell line (Buick et al., 1985), LOH (Yu et al., 1999). Thus, with the inclusion of the the SG10G cell line (Garzetti et al., 1992), the Xeroderma RNASE6PL gene, these results seem to indicate that Pigmentosum ®broblast cell line XP12ROSV, immortalized epigenetic mechanisms may play an important role in by SV40 (Simon et al., 1981) and the ovarian cancer cell lines ovarian tumorigenesis, by inactivation or downregula- OV1225, SKOV3, OVCR3, OVRS1000, SW626 and OV166 were cultured in DMEM/F12 medium (GIBCO ± BRL) tion of tumor suppressor genes. supplemented with 10% fetal calf serum. pRPcT1/H6c12T During the course of these experiments, when (pRPc) cells, derived from a tumor induced by the human/ RNASE6PL cDNA was transfected into HEY4 and mouse monochromosomic hybrid pRPcT1/H6, carrying a XP12ROSV cells, clear signs of senescence appeared in human chromosome 6 tagged with the neo gene (Gualandri et cell culture. The senescent phenotype was con®rmed by a al., 1994), were cultured in the same medium in the presence positive reaction for beta-galactosidase, an enzyme of 300 mg/ml of G418 (Boehringer, Mannheim). whose expression is speci®cally associated to the process of senescence (Dimri et al., 1995). Induction of Construction of plasmids and transfection of cells replicative senescence by RNASE6PL is particularly interesting in view of the fact that RNase activity pRPc cells were cotransfected with the RNASE6PL cDNA inserted in the plasmid pMT89 together with the pGK increases in plants during senescence and in human plasmid carrying the resistance to hygromicin. The HEY4 serum in the course of ageing (Schein, 1997; Francesconi and SG10G cells, which do not express the RNASE6PL gene et al., 1984). It is not clear at present why only a (see Results section), were transfected with the RNASE6PL proportion of cell clones, both in HEY4 and in cDNA inserted in the plasmid pcDNA3 (Invitrogen) which XP12ROSV cell cultures, displayed the senescence contains the neo gene and induces resistance to G418. In both phenotype. With the single exception of angiogenin, constructs, the RNase cDNA was directed by the cytomega- most known RNases (e.g. fungal RNase, BS-RNase, lovirus immediate early promoter. For transfection experi- onconase, EDN and the lectin RNases RCE and RJE) ments, the plasmid containing the RNASE6PL cDNA (6 mg are highly selective towards speci®c cell types (D'Alessio, per 106 cells) was transfected into pRPc, HEY4, SG10G, and 1993; Schein, 1997), suggesting that the other cells are XP12ROSV cells with the Superfect reagent (Quiagen), following the manufacturer's instructions. The clones, somehow protected from the inhibitory e€ect of RNases appearing after approximately two weeks of selection, were on cell growth. Therefore, it is possible that cell clones, harvested by trypsinization with glass cylinders and propa- not showing senescence after transfection of RNA- gated as mass cultures. SE6PL, derive from single cells developing protective mutations towards the activity of this RNase. Alter- Beta-galactosidase test staining natively, the RNASE6PL cDNA may be inactivated by mutation in transfected cell clones displaying a normal The test was performed directly in the culture ¯asks phenotype. Induction of senescence by RNASE6PL according to Dimri et al. (1995). makes this gene a good candidate for the senescence- inducing gene (SEN6) mapped at 6q27 (Banga et al., Thymidine incorporation test 1997), within the minimal region of LOH detected in Two6104 cells were cultured on glass dishes in 24-well plates ovarian cancer (Saito et al., 1992; Foulkes et al., 1993, in the presence of [3H]thymidine (1 mCi/ml). After 72 h, cells Cooke et al., 1996; Orphanos et al., 1995; Tibiletti et al., were extensively washed in PBS, dishes were recovered from 1996, 1998) or induction of senescence may simply be a the wells, resuspended in 1 ml of INSTA-GEL II PLUS phenotypic aspect of the tumor suppressor function (Packard) and the radioactivity was measured in a Packard displayed by RNASE6PL. The relation of RNASE6PL TRI-CARB 4530 beta counter. to SEN6 could be relevant to establish whether an immortalization step is an early event in the process of Tumorigenicity assays ovarian tumorigenesis. Indeed, the region where the RNASE6PL gene resides is consistently deleted not only To test for tumorigenicity in nude mice, cells were trypsinized, harvested and washed twice. Five6104 pRPc in benign ovarian tumors (Tibiletti et al., 1998) but also cells, 16105 HEY4 cells and 26106 SG10G cells were in other benign conditions such as parathyroid adeno- suspended in 0.1 ml of serum-free medium and inoculated s.c. mas (Tahara et al., 1996) and breast benign tumors into 3-week-old nude BALB/c mice. Animals were examined including ®broadenomas (Tibiletti et al., 2000). In for tumor formation for up to 6 months. After that time, if conclusion, RNASE6PL may represent the candidate tumors had not appeared, the cells were considered gene at 6q27 ful®lling both functions of inducing suppressed in tumorigenicity. senescence and of tumor suppressor gene in ovarian cancer. PCR and RT ± PCR Cells from each clone were harvested from 18 mm wells and suspended in 0.5 ml of culture medium. Twenty per cent of the suspension was directly used for PCR to con®rm the Materials and methods presence of the transfected cDNA, while the remaining 80% was used for RNA extraction followed by RT ± PCR. For Primary tumors, cell lines and culture conditions PCR reaction, the cell pellet was treated at 558C for 1 h in Fifty-®ve microdissected primary ovarian tumors, from stage 10 mM Tris pH 8.3, 1.25 mM MgCl2, 0.01% gelatine, 0.45% II to stage IV, were selected for mutational and expression Tween 20, 0.45% NP-40, 10 mg/ml proteinase K. After

Oncogene Tumor suppressor and senescence-inducing gene at 6q27 F Acquati et al 987 inactivation of proteinase K for 10 min at 958C, the reaction amplify unmethylated DNA) PCR conditions were as mixture was briefely centrifuged and 1 ml of the cell lysate follows: 948C 2 min, then 40 cycles at 928C30s,618C30s, was used for PCR analysis with T7 and Sp6 primers, 728C 60 s with primers 44bis-MetA and 44new-MetB; :948C designed on the arms of the expression vector. The conditions 2 min, then 40 cycles at 928C30s,598C30s,728C 60 s with of the reaction were: 948C for 30 s, 558C for 30 s, 728C for primers 44bis-UnmetA and 44new-UnmetB. As a control for 60 s, 30 cycles. For RT ± PCR analysis, total RNA was the speci®city of the primers, the same PCR reactions were extracted with the RNAwiz reagent (Ambion) following the carried out using DNA which had not been treated with manufacturer's instructions and reverse transcription was sodium bisul®te. carried out using the cDNA Synthesis System (Gibco ± BRL). GAPDH ampli®cation was performed to control cDNA PCR-single-strand conformation polymorphism (SSCP) synthesis. To discriminate from the endogenous RNASE6PL transcript, the expression of the transfected cDNA was tested DNA was prepared from 55 tumors and from the ovarian using the T7 primer from the vector and a speci®c primer for carcinoma cell line HEY4, using standard phenol-chloro- the RNASE6PL cDNA (5'-TTGGGCATCAATGCTTGG- phorm extraction. Primer pairs were designed in intronic 3'). cDNA ampli®cation was then carried out as described for regions ¯anking each exon and in the 5' and 3' untranslated the PCR reaction. regions of the RNASE6PL gene. Every single exon of the RNASE6PL gene was PCR ampli®ed (primer sequences and PCR conditions are available on request). SSCP analysis was RNASE6PL methylation status analysis by methylation specific performed on tumor DNAs as previously described (Varesco PCR (MSP) et al., 1993). MSP was carried out as described by Herman et al. (1996). Two mg of sodium bisul®te-treated genomic DNA from the samples of interest was puri®ed and used for MSP analysis using the following primers: 44bis-MetA: 5' TTTGCGT- Acknowledgments GGCGAAGGAACGTAGTCGTT-3' and 44new-MetB: 5'- R Taramelli was supported by grants from Associazione TACAACAACGACCACCGAATACGCCCG-3' (to amplify Italiana per la Ricerca sul Cancro (AIRC) and MURST methylated DNA); 44bis-UnmetA: 5'-GGTGTTTGTG- Co®n 1998. L Possati and G Barbanti-Brodano were TGGTGAAGGAATGTAGTTGTT-3', and 44new-UnmetB: supported by grants from AIRC and MURST ex 60% 5'-TCCACTACAACAACAACCACCAAATACACCC-3' (to and Co®n 1998.

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