Oncogene (1997) 15, 1289 ± 1294  1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00

Characterization of a highly conserved (OS4) ampli®ed with CDK4 in human sarcomas

Yan A Su, Margo M Lee, Carolyn M Hutter and Paul S Meltzer*

Laboratory of Cancer Genetics, National Research Institute, National Institutes of Health, Bethesda, Maryland 20892, USA

Ampli®cation and overexpression of involved in two novel genes (OS9 and OS4) (Su et al., 1994, 1996). cellular growth control occur frequently in human Recently, we have demonstrated that this amplicon is cancers. Here, we report characterization of the full derived from a gene-rich locus, encoding several length OS4 cDNA derived from 12q13-q15 (Su et al., additional ampli®ed cDNAs (Gracia et al., 1996). Proc. Natl. Acad. Sci. USA, 91: 9121 ± 9125, 1994), a Full characterization of ampli®ed DNA in tumors region frequently ampli®ed in sarcomas and brain requires mapping of the involved genomic segments to tumors. This cDNA consists of 4833 base pairs (bp) identify the genes contained in the core region which is encoding an open reading frame (ORF) of 283 amino consistently ampli®ed in multiple tumors. Expression acids. The ORF predicts a water-soluble acidic (pI 5.50) and functional studies are then required to interpret the polypeptide with a molecular weight of 31 759. Database relative importance of those genes which consistently searches revealed highly signi®cant similarity between contribute to the amplicon. Physical mapping studies OS4 and eight predicted from genomic have demonstrated that 12q13-q15 amplicons contain sequences of Caenorhabditis elegans, Schizosaccaharo- two separate core regions, a telomeric region contain- myces pombe, and Saccharomyces cerevisiae. Thus, OS4 ing MDM2 and a centromeric region containing CDK4 de®nes a novel evolutionarily conserved gene superfamily. (Elkahloun et al., 1996). These regions are separated by Northern and database analyses revealed OS4 transcripts over 1 Mb, and markers in variable portions of the in numerous human tissues demonstrating its ubiquitous intervening segment are frequently not ampli®ed. expression. We also observed overexpression of OS4 in Currently, because of their roles in regulating the cell three cancer cell lines with ampli®cation of this gene. cycle (MDM2 through p53, and CDK4 through pRb) Furthermore, we detected OS4 ampli®cation in 5/5 these genes are considered the most probable target primary sarcomas with known ampli®cation of the genes for 12q13-q15 ampli®cation. However, the CDK4 closely linked marker CDK4. These results demonstrate core region is quite gene dense, and several closely that the highly conserved OS4 gene is frequently included linked genes are typically co-ampli®ed (Berner et al., in the 12q13-q15 amplicon and may contribute to the 1996). We have been pursuing the complete character- development of a subset of sarcomas. ization of the CDK4 core region in order to evaluate the potential impact of the genes in this region on Keywords: neoplasm; genetics; 12q; tumor phenotype. OS4 which we originally identi®ed homogeneously staining region by chromosome microdissection and hybrid selection falls in the CDK4 core region, and we therefore pursued characterization of a full length cDNA clone. The 4833-bp OS4 cDNA encodes an ORF of 283 Introduction amino acid residues. Our analysis revealed OS4 expression in multiple di€erent human tissues and its In human cancers, gene ampli®cation is a common overexpression in three cancer cell lines with OS4 mechanism by which increased dosage of a gene leads ampli®cation. By Southern hybridization, we demon- to its overexpression. Ampli®cation of cellular onco- strated ampli®cation of OS4 in 5/5 primary sarcomas genes has been observed in tumor cell lines and with known CDK4 ampli®cation. Finally, database primary tumor tissues suggesting that overexpression searches demonstrate highly signi®cant similarity of of these genes provides tumor cells with a selective OS4 with eight genes in organisms as divergent as S. growth advantage in vitro and in vivo. The chromo- cerevisiae, strongly suggesting that these genes belong some 12q13-q15 region is frequently ampli®ed in to an evolutionarily conserved superfamily. human sarcomas and brain tumors (Smith et al., 1992; Reifenberger et al., 1994). Several genes have been previously mapped to this region, including a zinc Results ®nger (GLI [Roberts et al., 1989]), a member of the transmembrane four superfamily (SAS [Meltzer et OS4 cDNA sequence al., 1991; Jankowski et al., 1994]), a cyclin dependent kinase (CDK4 [Khatib et al., 1993]), a transcription The 4833-bp sequence of the full length OS4 cDNA factor (CHOP [Aman et al., 1992]), a modulator of p53 was determined from the concensus sequence of both (MDM2 [Oliner et al., 1992; Leach et al., 1993]), and strands of 57 plasmids containing overlapping OS4 cDNA inserts. The full length cDNA consists of three Correspondence: PS Meltzer segments: a 305-bp 5'-untranslated region (5'-UTR: 1 ± Received 14 March 1997; revised 20 May 1997; accepted 305), a 849-bp coding sequence (306 ± 1154), and a 20 May 1997 3679-bp 3'-untranslated region (3'-UTR: 1155 ± 4833). OS4 gene in human sarcomas YA Su et al 1290 The 305-bp 5'-UTR is GC-rich (72% GC content) and except that the levels was twofold lower in brain and contains several direct repeats. The OS4 ORF encodes lung, fourfold lower in liver, and more than fourfold 283 amino acids (Figure 1) with a predicted molecular higher in pancreas (Figure 3a). The levels of OS4 weight of 31.8 kD and pI 5.50. No hydrophobic transcripts in eight cancer cell lines were also similar segment longer than 10 amino acid residues was except for a somewhat lower level in the lung presented suggesting that the OS4 protein is probably carcinoma A549 (Figure 3b). water soluble. Database research revealed highly signi®cant simi- Over-expression of both OS4 and CDK4 in cancer cell larity of the OS4 ORF with eight ORFs deduced from lines with ampli®cation of these genes genomic sequences of C. elegans, S. pombe, and S. cerevisiae (Figure 2). (Figure 2a indicates the alignment Cancer cell lines with or without OS4 and CDK4 of OS4 with these ORFs underlining the presence of ampli®cation were used to study the expression of these three highly conserved regions (HC1, HC2 and HC3). two genes by Northern hybridization. The amplifica- HC1, HC2 and HC3 contains 76.5% (13/17), 56.8% tion of OS4 and CDK4 in several cancer cell lines (21/37), and 52.3% (23/44) identical residues, respec- including NGP-127 (neuroblastoma) (Su et al., 1994), tively, in six or more proteins of these nine ORFs OsA-Cl (osteosarcoma) (Su et al., 1994), and RMS-13 (Figure 2b). Out of these nine ORFs, four are from S. (rhabdomyosarcoma) (Elkahloun et al., 1996) have cerevisiae (two acidic, one neutral, and one basic), and been previously reported. The levels of OS4 and CDK4 three ORFs are from C. elegans (acidic, neutral, and transcripts from these ampli®ed cell lines were basic). The human and S. pombe ORFs are acidic. In compared with three normal tissues (spleen, pancreas, addition, two of the predicted polypeptides (F45E12.1 and skeletal muscle) and with the osteosarcoma cell and YHR004c) contain a hydrophobic segment (25 to lines U-2 OS (Ponten and Saksela, 1967) and HOS 35 residues) within the amino terminal region (Rhim et al., 1975) which had no Southern-detectable suggesting possible membrane anchorage. ampli®cation of OS4 and CDK4 (data not shown). The Although comparison of the OS4 cDNA sequence results from Northern hybridization demonstrated with the current GenBank database using the BLAST overexpression of both OS4 and CDK4 in the programs (Altschul et al., 1990) revealed no signi®cant ampli®ed cancer cell lines NGP-127, OsA-Cl, and identity to any gene with known function, a RMS-13 (Figure 4). After normalization relative to b- comparison with the EST (expressed sequence tag) actin, expression of OS4 and CDK4 was estimated by database revealed highly signi®cant alignments with densitometry to be at least tenfold higher in the over 100 ESTs (P-values: 10710 to 107198). These ESTs ampli®ed cell lines than in the non-ampli®ed lines were derived from 22 di€erent adult, infant, and fetal and the normal tissues. tissues, indicating ubiquitous expression of OS4.In addition, several highly similar ESTs from mouse and Ampli®cation of OS4 and CDK4 in primary sarcomas rat were also identi®ed by sequence alignment (data not shown). Since OS4 and CDK4 are linked within 60 kb (Elkahloun et al., 1996), Southern analysis to evaluate co-ampli®cation of OS4 with CDK4 was performed on Ubiquitous expression of OS4 DNA samples isolated from ®ve di€erent human To examine the expression of OS4, Northern blots primary sarcomas with CDK4 ampli®cation previously containing poly(A)+ RNA from adult normal tissues identi®ed by screening with a CDK4 probe. The result and cancer cell lines were hybridized with the OS4 demonstrated the ampli®cation of OS4 in all ®ve probe. The result demonstrated similar levels of OS4 primary sarcomas tested consistent with its presence transcripts in most of 16 normal tissues examined within the core region of ampli®cation (Figure 5).

Figure 1 The predicted 283 amino acid sequence of the OS4 ORF. The underlined residues in the amino acid sequence correspond to three highly conserved regions illustrated in Figure 2a and 2b. The map illustrates the chromosomal order and approximate positions of OS4 and three closely linked and frequently co-ampli®ed genes (CDK4, SAS, and OS9 [Elkahloun et al., 1996]). The 4833 bp full length OS4 cDNA sequence was submitted to GenBank (accession number: AF000152) OS4 gene in human sarcomas YA Su et al 1291

Figure 2 Amino acid sequence alignments of OS4 with other ORFs. (a) An overview of the alignments of highly conserved regions (HC1, HC2 and HC3 [dark shading]) and moderately conserved regions (light shading) in the nine ORFs. Ce1, B0379.4 of C. elegans (GenBank: 1865728); Sp1, SPAC2F7.02c of S. pombe, (Genbank: 1175367); Sc1, YLR109w of S. cerevisiae (GenBank: 1360322); Sc2, YLL010c of S. cerevisiae (GenBank 1360157); Ce2, F45E12.1 of C. elegans (GenBank: 868266); Sc3, YHR004c of S. cerevisiae (GenBank: 731630); Sc4, LPE7w of S. cerevisiae (GenBank: 1079670); Ce3, T21C9.12 of C. elegans (GenBank: 1313954). The numbers in parentheses indicate the P-value of the alignment between the corresponding ORF and OS4. The open and shaded bars represent ORFs. The numbers of amino acid residues (AAR) and the isoeletric points (pI) of each ORF are indicated. (b) Amino acid sequence alignments of HC1, HC2, and HC3 among the nine ORFs. The numbers indicate a position of the segments in each ORF. Identical residues in ®ve or more ORFs are shaded. In the consensus sequence, the capital letters indicate the residues identical in seven or more proteins, and the lower case letters indicate residues identical in ®ve and six proteins. Protein alignments were created by the Lipman-Pearson program in DNASTAR software (version 1.58; DNASTAR Inc.)

a b Spleen Thymus Prostate Testis Ovary Intestine Colon W.B.C Heart Brain Placenta Lung Liver Muscle Kidney Pancreas 7.5 — HL-60 Hela K562 MOLT-4 Raji SW480 A549 G361

4.4 — — OS-4

2.4 — — β-actin

1.4 — Figure 3 Northern analysis of OS4. The OS4 probe detects 4.8-kb transcripts in 16 human normal tissues (a) and eight cancer cell lines (b). (a) The levels of OS4 poly(A)+ RNA were twofold lower in brain and lung, fourfold lower in liver, and more than fourfold higher in pancreas, than in the remaining normal tissues. (b) OS4 transcripts was detected in these cancer cell lines at levels comparable to those observed in normal tissues. The message level was some lower in A549 relative to the other lines tested. The numbers indicate molecular markers (kb). The human b-actin probe was used as a control for loading error. The weak and strong bands below b-actin in the lanes of prostate, colon, heart, and skeletal muscle are isoforms of b-actin mRNA. W.B.C., white blood cells; muscle, skeletal muscle; HL-60: promyelocytic leukemia HL-60; Hela: HeLa cell S3; K562: chronic myelogenous leukemia K562; MOLT-4: lymphoblastic leukemia MOLT-4; Raji: Burkitt's lymphoma Raji; SW480: colorectal adenocarcinoma SW480; A549: lung carcinoma A549; G361: melanoma G361 OS4 gene in human sarcomas YA Su et al 1292 family. Overexpression of OS4 and the closely linked gene CDK4 was revealed in three cancer cell lines with ampli®cation of these genes. In addition, we demon-

Spleen Pancreas Muscle NGP-127 RMS-13 OsA-CI U-2 OS HOS strated co-ampli®cation of OS4 with CDK4 in 5/5 primary human sarcomas with known CDK4 amplifi- cation. 7.5 — OS-4 The full length OS4 cDNA consists of 4833-bp nucleotides which is consistent with Northern analysis detecting a message of approximately 4.8 kb in all 16 human normal tissues and 13 cancer cell lines 4.4 — CDK4 examined. Interestingly, the GC-rich 305-bp 5'-UTR contains a multiple direct repeat clusters, suggesting a possible role in regulation of either transcription or translation of the OS4 gene. Translation of the OS4 2.4 — ORF predicts a water-soluble acidic polypeptide. The β -actin presence of OS4 transcripts in several dozens of 1.4 — di€erent human tissues including normal tissues (adult, infant, and fetal), tumor tissues, cancer cell Figure 4 Overexpression of OS4 and CDK4 in cancer cell lines with ampli®cation of these genes. After normalization relative to lines, and a multiple sclerosis lesion demonstrates its b-actin, expression of OS4 and CDK4 was estimated by ubiquitous expression. Although its function has yet to densitometry to be more than tenfold higher in the cell lines, be determined, such a broad spectrum of expression NGP-127, RMS-13, and OsA-Cl with ampli®cation of these two suggests that OS4 has an essential role. genes than the non-ampli®ed lines, U-2 OS and HOS, and the normal tissues, spleen, pancreas, and skeletal muscle. NGP-127: Using the predicted OS4 protein, we identi®ed eight neuroblastoma (Schwab et al., 1983); RMS-13: rhabdomyosarco- genes from genomic sequences of C. elegans, S. pombe, ma (Roberts et al., 1989; OsA-C1: osteosarcoma (Roberts et al., and S. cerevisiae sharing highly conserved sequences 1989); U-2 OS: osteosarcoma (Ponten and Saksela, 1967); HOS: (HC1, HC2, and HC3). According to the predicted osteosarcoma (Rhim et al., 1975) isoelectric point, the nine proteins can be divided into three subgroups: acidic (OS4, B0379.4, SPAC2F7.02c, YLR019w, and YLL010c), basic (F45E12.1 and YHR004c), and neutral (LPE7w and T21C9.12). Because of the potential transmembrane domain in the amino terminal region of F45E12.1 and YHR004c, Placenta T1 T2 T3 T4 T5 these two peptides are possibly membrane-associated. The highly signi®cant sequence similarity in these nine OS-4 predicted proteins strongly suggests that they share the 9.4 — domains with common function. Currently, this super- family of proteins contains four members from S. cerevisiae and three members from C. elegans.We expect more members to be identi®ed from other 9.4 — CDK4 eukaryotes. In fact, several human ESTs aligned with the OS4 cDNA appear to represent additional human genes in the OS4 family. 6.6 — The potential biological role of OS4 in relation to β 12q13-15 ampli®cation must be considered in the -actin context of physical mapping studies of this amplicon. 4.4 — We have previously demonstrated that there are two distinct core regions of ampli®cation separated by Figure 5 Southern detection of OS4 and CDK4 ampli®cation in several megabases. Although each of these segments human sarcomas. After normalization relative to b-actin, in comparison to placental DNA, both OS4 and CDK4 probes can be ampli®ed independently, they are most detected greater than ®vefold intensity in T1, T2, T3, and T5, and frequently co-ampli®ed, and the intervening region greater than twofold ampli®cation in T4, estimated by remains single copy. This pattern has been observed in densitometry. T1: pleomorphic sarcoma 269; T2: malignant both sarcomas and brain tumors (Berner et al., 1996; ®brous histocytoma 19A; T3: osteosarcoma C128; T4: osteosar- Reifenberger et al., 1996. The more telomeric of these coma C206; T5: malignant schwannoma C080. 10 mgofDNA was digested with HindIII, size-fractionated on an agarose gel, two ampli®ed segments includes the MDM2 gene and transferred to a nylon membrane for hybridization which functions as a negative regulator of p53.At the present time, no additional ampli®ed genes have been identi®ed in the MDM2 core region. The second region of ampli®cation is located several Mb centro- Discussion meric to MDM2 and is approximately 100 kb in size. This amplicon contains a very tightly linked cluster of In this report, we have presented the features of the genes including CDK4, OS4, SAS, and OS9 as well as OS4 cDNA and ORF, and its ubiquitous expression in several other cDNAs (Elkalhoun et al., 1997). CDK4 is human normal tissues and cancer cell lines. Our an excellent candidate target gene for this amplicon in homology analysis showed that OS4 and the related view of its role in promoting the G1 to S phase genes from C. elegans, S. pombe,andS. cerevisiae, transition through phosphorylation of the retinoblas- probably belong to an evolutionarily conserved super- toma protein. However, even if CDK4 is accepted as OS4 gene in human sarcomas YA Su et al 1293 the primary target of this ampli®cation unit, it is (Sambrook et al., 1989). Thirty-nine plaques were picked dicult to exclude a contribution to the transformed following hybridization with the OS4 probe and converted phenotype from overexpression of nearby ¯anking to pBluescript plasmids containing OS4 cDNA inserts genes such as OS4. It thus remains necessary to according to the manufacturer's protocol (Stratagene). A characterize each gene in the core of the ampli®cation total of 13 plasmids were used for determination of the 3'- most 1.2-kb sequence of the OS4 cDNA. Using this unit with regard to expression, sequence and function. sequence, 13 plasmids were isolated from a spleen cDNA In this regard OS4 is intriguing because of its library using the GeneTrapper method (GIBCO BRL). remarkably high degree of sequence conservation. These plasmids resulted in extension of the cDNA sequence Although its pattern of expression appears to be to 2.8 kb. Using previously described 5' RACE methods ubiquitous, the levels of expression which we observed (Su et al., 1996), we generated 31 plasmids containing in association with gene ampli®cation are signi®cantly overlapping fragments of the 5'-most 2.5-kb OS4 cDNA. higher than those observed in normal tissues and might well result in a phenotypic e€ect. Because of these Nucleotide sequence analysis considerations, further functional studies of OS4 appear justi®ed to determine to what extent its Both strands of a total of 57 plasmids containing overlapping fragments of the full length OS4 cDNA were overexpression may impact on the biological proper- sequenced by automated ¯uorescence sequencing. Multiple ties of tumor cells bearing ampli®cation of this region. sequence alignments and consensus determinations were performed with DNASTAR software (version 1.58; DNASTAR Inc.). The current sequence databases were searched by the BLASTN, BLASTP, and FASTA Materials and methods programs (Altschul et al., 1990). Cell culture and isolation of RNA and cDNA Northern and Southern hybridization The human cancer cell lines, OsA-Cl (osteosarcoma) (Roberts et al., 1989), RMS-13 (rhabdomyosarcoma) Human b-actin and CDK4 probes were described (Roberts et al., 1989), NGP-127 (neuroblastoma) (Schwab previously (Su et al., 1996). The OS4 probewasa2.7kb et al., 1983), HOS (osteosarcoma) (Rhim et al., 1975), U-2 OS4 cDNA. The probes were labeled by random priming OS (osteosarcoma) (Ponten and Saksela, 1967), were (Feinberg et al., 1983). Multiple tissue Northern mem- cultured in RPMI 1640 medium (GIBCO BRL) supple- branes were obtained from Clontech. Northern and mented with 10% fetal bovine serum (HyClone) and 2 mM Southern hybridizations were performed by standard L-glutamine (GIBCO BRL). OsA-Cl and RMS13 were methods (Sambrook et al., 1989; Brown, 1993). Filters kindly provided by Tom Look, St. Jude Children's were washed to a stringency of 0.16SSC at 428Cand Research Hospital, Memphis, Tennessee. NGP-127 was exposed to Kodak X-omat AR autoradiography ®lm at kindly provided by Garrett Brodeur, Children's Hospital of 7808C for various periods from 2 h to 3 days. Band Philadelphia. The remaining cell lines were obtained from intensities were measured by densitometry and analysed the American Type Culture Collection (ATCC). using IP Lab Spectrum software (Signal Analytics Co., Sarcoma tumor tissue was provided by the Cooperative Vienna, Virginia). Human Tissue Network (CHTN), Birmingham, Alabama. DNA was isolated by standard methods (Sambrook et al., 1989). Poly(A)+ RNA was puri®ed using the FastTrack kit 2.0 (Invitrogen). Poly(A)+ RNA from spleen, pancreas, and Abbreviations skeletal muscle were obtained from Clontech. ORF, open reading frame; hsr, homogeneously staining region; CDK4, cyclin-dependent kinase 4; pRb, retinoblas- toma protein; EST, expressed sequence tag; bp, base pairs. Generation of plasmids containing the OS4 cDNA The cDNA library (in bacteriophage l ZAPII) highly enriched for transcribed sequences from the 12q hsr was previously described (Su et al., 1994). The library was Acknowledgements plated at a density of 628 plaque forming units (pfu) per We thank Ping He, Xiaoqing He, and Robert L Walker for 150-mm petri dish in E. coli XL1-blue cells. Plaque lifts technique assistance and Darryl Leja for help with graphic and hybridizations were performed by standard methods illustration.

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