Oncogene (1998) 17, 1019 ± 1026 1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 http://www.stockton-press.co.uk/onc Co-ampli®cation of a novel cyclophilin-like gene (PPIE) with L-myc in small cell lung cancer cell lines Ju-Ock Kim1,3, Marion M Nau1, Kathrine A Allikian1, Tomi P MaÈ kelaÈ 2, Kari Alitalo2, Bruce E Johnson1 and Michael J Kelley1 1Lung Cancer Biology Section, Medicine Branch, Division of Clinical Sciences, National Cancer Institute, Bethesda, Maryland, 20889-5105 USA and 2Laboratory of Cancer Biology, Department of Virology and Pathology, University of Helsinki, 00290 Helsinki, Finland Speci®c genetic alterations aecting proto-oncogenes of myc,N-myc, and L-myc) occurs in lung cancer both in the myc gene family are frequently detected in human the presence and absence of other detectable genetic lung cancer. Among 11 SCLC cell lines with L-myc gene alterations such as gene ampli®cation and genomic ampli®cation, four were found to have alteration of the rearrangement. In patients with small cell lung cancer RLF gene by Southern blot and RT ± PCR analyses. (SCLC), DNA ampli®cation of c-myc is more common One cell line, NCI-H378, contained aberrantly-sized L- in tumor cell lines established from treated than myc-hybridizing bands by Southern and Northern blot untreated patients' tumors (Johnson et al., 1996). hybridization but had no alteration of RLF. Some L- DNA ampli®cation of c-myc is also associated with myc-hybridizing cDNAs from NCI-H378 contained a the variant form of SCLC cell lines (Little et al., 1983), novel sequence with close homology to the cyclophilins which typically have greater cloning eciency in soft joined to antisense L-myc exon 2 sequence. Full length agar, shorter doubling time in vitro, altered in vitro and cDNAs isolated from human skeletal muscle containing in vivo morphology, and lower expression of markers only the novel sequence identify open reading frames of of neuroendocrine dierentiation compared to the 301 and 296 amino acids and dier in the C-terminal more common classic SCLC cell lines (Carney et al., region by 22 and 17 amino acids. This gene, tentatively 1985; Gazdar et al., 1985). In contrast, tumors with L- named PPIE (peptidyl-prolyl cis-trans isomerase E), has myc ampli®cation are most commonly of the classic 83% amino acid identity with the central conserved morphology (Johnson et al., 1996). In patients with region of cyclophilin A, is evolutionarily conserved by previously treated SCLC, the presence of c-myc DNA Southern blot, and exhibits dierential tissue expression ampli®cation is associated with shortened survival with highest levels found in muscle and brain. Co- (Johnson et al., 1996). ampli®cation of PPIE was observed in seven of eleven L- The human L-myc gene (also known as MYCL1) myc ampli®ed cell lines. Analysis of radiation hybrids was initially identi®ed in SCLC cell lines and tumors suggests that the gene order is RLF-PPIE-L-myc on because of DNA ampli®cation of sequences homo- chromosome 1p and pulse-®eld gel electrophoresis logous to but dierent from the human c-myc and N- localizes all three genes to an 800 megabase Mlu I myc genes (Nau et al., 1985) and consists of three fragment. The prognostic and functional consequences of exons spanning approximately 6.6 kb (Kaye et al., PPIE gene ampli®cation in SCLC can now be 1988) on chromosome 1p32 (Nau et al., 1985). L-myc determined. DNA ampli®cation is found in 18% of SCLC cell lines and 12% of SCLC tumors (Johnson, 1996). The Keywords: gene ampli®cation; L-myc; cyclophilin; small various sized L-myc transcripts, including 3.9 kb, cell lung cancer; tumor cell line; alternative splicing 3.6 kb, and smaller sized species, result from alter- native transcription initiation and termination sites (Kaye et al., 1988), and all include exons 2 and 3 and a a predicted 364 amino acid protein (De Greve et al., Introduction 1988). Alternative translation initiation from a CTG start site in intron 1 and post-translational modifica- Speci®c chromosomal alterations including transloca- tion results in proteins with apparent molecular weight tions, inversions, deletions, and DNA ampli®cation of 60 to 66 kDa (Dosaka-Akita et al., 1991). have been identi®ed in a large number of tumor types. Observation of an abnormally-sized protein on L- Activation of proto-oncogenes of the myc family (c- Myc immunoblots of SCLC cell lines led to identifica- tion of the RLF (rearranged L-myc fusion) gene (MaÈ kelaÈ et al., 1991b), which is 310 kb centromeric to L-myc (Hellsten et al., 1995). Juxtaposition of the RLF and L-myc genes in SCLC cells results in Correspondence: M Kelley expression of a chimeric RLF-L-myc transcript 3Current address: Department of Internal Medicine; Chungnam National University Hospital; 640 Daesadong Jungku, Taejon, (MaÈ kelaÈ et al., 1991b). The resulting chimeric RLF- Korea 301-040 L-Myc protein, like the wild-type L-Myc protein, The opinions or assertions contained herein are the private views of inhibits dierentiation in murine embryonic stem cells the authors and are not to be construed as ocial or as re¯ecting the (MacLean-Hunter et al., 1994). Characterization of 126 views of the Department of the Navy or the Department of Defense. SCLC cell lines at NCI has identi®ed 11 (9%) with L- This is a U.S. government work. There are no restrictions on its use. Received 9 January 1998; revised 25 March 1998; accepted 26 March myc ampli®cation (Johnson et al., 1996). We studied 1998 these eleven L-myc-ampli®ed cell lines and have Co-amplification of PPIE with L-myc in SCLC J-O Kim et al 1020 identi®ed a novel cyclophilin-like gene located at ampli®ed cell lines was performed using probes from chromosome 1p32. Co-ampli®cation on this cyclophi- L-myc exon 2 and exon 3 (Figure 1). The abundant L- lin-like gene with L-myc in SCLC cell lines occurs more myc transcript observed in NCI-H378 by hybridization commonly than co-ampli®cation of RLF with L-myc with either an exon 2 or exon 3 probe was slightly and can result in expression of chimeric transcripts. smaller (*3.6 kb) compared with the expected bands (*3.9 and 3.7 kb) (Figure 1). Expected-sized tran- scripts were observed in the other 10 L-myc ampli®ed cell lines. In addition, aberrantly-sized transcripts of Results approximately 3.2 kb were observed in both NCI-H889 and NCI-H1836 (Figure 1). No L-myc mRNA was Characterization of RLF in L-myc ampli®ed cell lines detected in NCI-H289, suggesting that L-myc gene Southern blot analysis of SstI-digested genomic DNA ampli®cation is not invariably associated with over- from the L-myc-ampli®ed cell line, NCI-H378, has expression of mRNA. previously been found to detect a slightly smaller DNA To determine if RLF rearrangement was responsible band (3.5 kb) compared to normal genomic DNA for these aberrantly-sized transcripts, Northern blot when hybridized with an exon 2 L-myc probe (Johnson analysis was performed using a probe containing RLF et al., 1987; Nau et al., 1985). To determine if aberrant- exon 1 and 2 sequence. This analysis detected sized transcripts are present in this cell line, Northern transcripts only in NCI-H889 and NCI-H1836 blot analysis of NCI-H378 and 10 additional L-myc- (Figure 1). RT ± PCR using an L-myc exon 3-speci®c oligonucleotide (MK60; Table 1) as primer for the RT reaction and an oligonucleotide from RLF exon 1 (MK59; Table 1) and L-myc exon 2 (MK53; Table 1) as PCR primers was also performed on the eleven L- myc ampli®ed cell lines. Using this technique, chimeric RLF-L-myc transcripts were detected in three of eleven L-myc-ampli®ed cell lines (NCI-H889, NCI-H1836, and NCI-H1994; data not shown) and con®rmed by direct sequencing of the PCR products to contain RLF exon 1 and L-myc exon 2 sequence (MaÈ kelaÈ et al., 1991a). However, no RLF-L-myc chimeric transcript was detected in NCI-H378. Therefore, the aberrantly- sized L-myc transcript in NCI-H378 did not result from a RLF-L-myc gene rearrangement. To determine the frequency of RLF gene amplifica- tion in the NCI L-myc-ampli®ed cell lines, Southern blot analysis of SstI-digested genomic DNA from SCLC cell lines using a full length RLF cDNA as probe was performed (Figure 2). Four bands [*15 kb, Figure 1 Northern blot analysis of L-myc ampli®ed cell lines 12 kb, 5 kb (doublet), and 3.8 kb] were detected in all using probes from L-myc (exon 2 and 3) and RLF. Approximately 20 mg of total cellular RNA was subjected to Northern blot eleven L-myc-ampli®ed cell lines, as well as in a human analysis as described in Materials and methods and hybridized B lymphoblastoid cell line (NCI-B1184); a L-myc- sequentially with 32P-radiolabeled probes from exon 2 and 3 of L- overexpressing, non-L-myc ampli®ed cell line (NCI- myc and an RLF probe containing exon 1 and 2 sequence (300 bp H209); a c-myc-ampli®ed cell line (NCI-H211); a non- EcoRI-HindIII fragment of plasmid a8-1.2). A rat GAPDH L-myc overexpressing, non-MYC ampli®ed cell line cDNA fragment served as a control probe (lower panel). The B lymphoblastoid cell line, B1184, was included as a negative (NCI-H345); and an N-myc-ampli®ed cell line (NCI- control H526) (Figure 2a).