Proc. Natl. Acad. Sci. USAVol. 83, pp. 2556-2560, April 1986Genetics

Localization of the human pim oncogene (PIM) to a region ofchromosome 6 involved in translocations in acute leukemias

(oncogene activation/chromosome translocations/human leukemias)

LALITHA NAGARAJAN, ELAINE LoulE, YOSHIHIDE TsuJIMOTO, ABBAS AR-RUSHDI, KAY HUEBNER,AND CARLO M. CROCEThe Wistar Institute, 36th at Spruce Street, Philadelphia, PA 19104

Communicated by Peter C. Nowell, December 9, 1985

ABSTRACT The human homolog, hpim, of the murinepim-1 gene, which is activated in murine T-cell lymphomas byinsertion of retrovirus proviral genomes in the pim-1 region,has been molecularly cloned; the cloned probe has been used tomap the hpim locus to human chromosome region 6p2i bysomatic cell hybrid analysis and chromosomal in situ hybrid-ization. The hpim gene is expressed as a 3.2-kilobase mRNA invarious human cell lines of hematopoietic lineage, most dra-matically in the K562 erythroleukemia cell line, which containsa cytogenetically demonstrable rearrangement in the 6p2lregion. A characteristic chromosome anomaly, a reciprocaltranslocation t(6;9)(p21;q33), has been described in myeloidleukemias and could involve the hpim gene.

In rodents and chickens, oncogene activation and neoplasticdisease can result from chromosomal integration of anonacute transforming virus near potentially transforminghost genes. Tumor induction by this type of insertionalmutagenesis was first demonstrated in chickens, in which themajority of bursal lymphomas induced by avian leukosisvirus contain proviral integrations near the c-myc oncogene(1, 2). The mouse mammary tumor virus proviral genome hasbeen observed to be integrated into distinct chromosomaldomains in virally induced mammary carcinoma (3-5), and inMoloney murine leukemia virus (MuLV)-induced lymph-omas in rats, a MuLV provirus is frequently integrated indistinct chromosomal domains (6, 7). Recently, a number ofmink cell focus-forming (MCF) proviruses were molecularlycloned from mouse lymphoma DNA, and flanking-regionprobes were used to detect common integration regions inother MuLV-induced lymphomas. One clone revealed vari-ations in the molecular organization of the correspondingregion in other lymphomas. This murine genetic locus, pim-1,was rearranged in more than 25% of T-cell lymphomas thatwere derived from different mouse strains and with variousMuLVs (8, 9).The murine pim-1 gene is expressed as a 2.8-kilobase (kb)

mRNA at low levels in normal lymphoid tissues (10). Proviralintegration in the pim-1 locus in MuLV-induced T-celllymphomas has been reported to be associated with thepresence of enhanced levels of pim-1 mRNA, which mayrange in size from 2.0 to 2.6 kb depending on the site ofintegration of the provirus (10). Other investigations haveobserved pim-J expression in replicating murine T cells butnot in various murine T-cell neoplasms (11).Because of our interest in oncogenes and potential onco-

genes involved in human B- and T-cell neoplasia, we haveisolated human genomic sequences homologous to themurine pim-1 gene. To assess involvement of the human pimlocus (PIM, hereafter referred to as hpim) in human

neoplastic disease, we have determined the location of thehpim gene in the human genome and have determined thatour genomic clones do indeed represent an active gene invarious cell lines of hematopoietic origin.

MATERIALS AND METHODS

hpim Genomic Clones. The mouse pim-J probe, a 1-kbBamHI fragment of the murine pim-1 genomic clone (8),hybridized to a human 3.6-kb EcoRI fragment and to a human14-kb Bcl I fragment. Bcl I-digested, size-selected 14-kb DNAfrom cell line 380 (12) was cloned in EMBL3A X phage vector.A 14-kb clone from the 380 genomic library, hpim5, thathybridized with the mouse pim-1 probe is shown in Fig. 1.The human 3.6-kb, repeat-free EcoRI fragment (phpim5Rl)contained within this 14-kb X phage recombinant was labeledby nick-translation and used as probe in DNA, RNA, andchromosomal in situ hybridization experiments.

Cells. Isolation, propagation, and characterization of pa-rental cells and somatic-cell hybrids used in this study havebeen described (13-18). All hybrids were characterized forexpression ofenzyme markers assigned to each of the humanchromosomes (13, 14). Some hybrid clones were karyotypedby trypsin/Giemsa and/or G-11 banding methods as de-scribed (14). In addition, the presence of specific humanchromosomes in many ofthe mouse-human hybrids has beenconfirmed by DNA hybridization using probes for genesassigned to specific human chromosomes (13-18).

Southern Blot Analysis. DNAs from human peripheralblood lymphocytes or leukemia cells, mouse cell lines, andmouse-human hybrid cell lines were prepared by cell lysis,proteinase K digestion, phenol extraction, and ethanol pre-cipitation. Cellular DNAs were digested with an excess ofappropriate restriction enzymes, electrophoresed in 0.8%agarose gels, and transferred to nitrocellulose or nylon filtersas described by Southern (19). Hybridization with 32P-labelednick-translated probe was carried out at 680C in 6x SSC/2XDenhardt's solution containing 0.2 mg of sonicated salmonspermDNA per ml. (1 x SSC is 0.15M NaCl/0.015M sodiumcitrate, pH 7.0; lx Denhardt's solution is 0.02% bovineserum albumin/0.02% polyvinylpyrrolidone/0.02% Ficoll.)Filters were washed in 0.1x SSC/0.1% NaDodSO4 at 680C.Hybridized filters were exposed to KodakXAR film at -700Cin the presence of an intensifying screen for 12-24 hr.RNA Blot Analysis. For analysis of hpim transcripts in

human cell lines, total cytoplasmic RNA was extracted bysubjecting cells to a brief hypotonic treatment, lysis in 0.5%Nonidet P-40 in the presence of N-dodecanoylsarcosine, andcentrifugation of the lysate through a cushion of 5.7 M CsCl,as described (20). RNA was denatured in 50% (vol/vol)formamide/2.2 M formaldehyde, electrophoresed in 1%agarose, and transferred to nitrocellulose membrane. Prehy-

Abbreviation: kb, kilobase(s).

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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B Bpim-J probe

II I

HB BI I I I I I I I 1Xhpim5E B B H H E H H HC

I | phpim5Rl1 kb

FIG. 1. Restriction map of the hpim locus. BCl I-digested line 380 cellular DNA in the size range of 14 kb was ligated to BamHI-digestedEMBL3A phage vector and packaged in vitro. Approximately 90,000 independent recombinant bacteriophage plaques were tested forhybridization to the 1.0-kb murine pim-) genomic clone. A restriction map of the single recombinant clone obtained is shown; the regions ofhybridization to the mouse probe and the human genomic fragment phpim5R1 used in this study are also depicted. B, BamHI; C, Bcl I; H,HindIII; E, EcoRI.

bridization and hybridization to 32P-labeled nick-translatedphpim5R1 were performed as described by Thomas (21).In Situ Hybridization. Metaphase spreads were prepared

from normal human male lymphocyte cultures stimulatedwith phytohemagglutinin for 72 hr. The phpim5R1 3.6-kbDNA insert was excised from the 14-kb X phage recombinantclone hpim5 and nick-translated with [3H]dCTP (58.3Ci/mmol; 1 Ci = 37 GBq; New England Nuclear), [3H]dGTP(25 Ci/mmol), [3H]dTTP (96 Ci/mmol), and [3H]dATP (50Ci/mmol). The techniques used for chromosomal in situhybridization were essentially as described by Harper andSaunders (22). Chromosome preparations were treated withpancreatic RNase A (Sigma) and then denatured in 70%oformamide in 2x SCC (pH 7.0) at 70'C for 2 min. Thechromosome preparations were then hybridized with 3H-labeled probe phpim5Rl DNA (specific activity, 2 x 107cpm/Ag) at a concentration of 70-100 ng/ml in 50%formamide/2x SSC/10% dextran sulfate (Pharmacia), pH7.0, for 20 hr at 370C. A 200-fold excess of sonicated salmonsperm DNA was included as carrier. Slides were thoroughlyrinsed in 50% formamide, 2 x SSC at 390C, exposed to KodakNTB2 nuclear track emulsion for 16 days at 40C, anddeveloped with Kodak Dektol at 15'C. The chromosomeswere then G-banded, essentially as described by Cannizzaroand Emanuel (23), with a mixture of 6 parts borate buffer (50mM Na2SO4/2.5 mM Na2B407, pH 9.2) to 1 part Wright/Giemsa stain solution (2.4 g of Wright stain and 1.4 g ofGiemsa stain per liter in methanol).

translocation chromosome. The hybrid retaining the 17p+chromosome carrying the region 6p21-6pter was previouslyfound to retain the gene for human glyoxalase (GLO) and thehuman histocompatibility locus (HLA) (24); the counterse-lected hybrid had lost the 17p+ chromosome as well as theGLO and HLA loci (25).As shown in Fig. 3, the hybrid retaining the 17p+ chromo-

some retained the human hpim gene, while the hybridcounterselected with bromodeoxyuridine had lost both the17p+ and the hpim locus. Thus, the hpim gene is located on

region 6p21-6pter.Regional Localization of hpim on Chromosome 6 by in situ

Hybridization. To locate the hpim locus more precisely on theshort arm of chromosome 6, we performed in situ hybridiza-tion of the 3H-labeled phpim5R1 probe to normal humanmetaphases. As shown in Fig. 4, the phpim5R1 probehybridized preferentially to region 6p12-p21. Fig. 4 Uppershows a representative human metaphase with an autoradio-graphic grain over the short arm of chromosome 6. Fig. 4

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19kp 4

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RESULTS

Chromosome Localization of hpim by Somatic Cell HybridAnalysis. In order to assign the hpim gene to a specific humanchromosome, a prerequisite for determination of possiblehpim involvement in human neoplasia, DNA from a panel ofwell-characterized rodent-human hybrid cells containingdifferent overlapping subsets of human chromosomes wastested for the presence of sequences homologous to the hpimprobe phpim5R1 (Fig. 1). As shown in Fig. 2 and Table 1, thepresence of the hpim-specific 3.6-kb band segregated con-cordantly with the presence of human chromosome 6 in thehybrid clones. These results indicate that the hpim locus is onhuman chromosome 6 (Table 1).To regionally localize the hpim gene on chromosome 6, we

have also hybridized the phpim5Rl probe to EcoRI-digestedDNA from two additional mouse-human hybrids: a mouse-human hybrid retaining a t(6;17)(6pter-6p21::17pl3-17qter)translocation chromosome (17p+) in the absence of the intactchromosome 6 and the deleted chromosome 6 (6p21-6qter)(24, 25); and the same hybrid clone after counterselection inmedium containing 5-bromodeoxyuridine, which selects forhybrid cells that have lost the t(6;17)(6pter-6p21: :17pl3-17qter)

FIG. 2. The human pim-1 homolog, hpim, maps to chromosome6. Southern blot analysis of DNA (10 gg per lane) from a mouse cellline (lane 1); human peripheral blood lymphocytes (lane 2); andmouse-human hybrids retaining the following human chromosomes:chromosomes 6, 7, 17q, 21 (lane 3); chromosome 7 (lane 4);chromosomes 1, 3-10, 13, 14, 16, 17, 20, 22, X (lane 5); chromosomes1, 3-10, 13, 14, 17, 20, 22, X (lane 6); chromosomes 3-5, 14, 15, 17,20, 22, X (lane 7); chromosomes 5, 8, 14, 15, 17-19, 21, 22, X (lane

8); chromosomes 4, 6, 7, 13, 16-18, 20 (lane 9); chromosomes 4, 7,8, 13, 15, 17, 18, 20, X (lane 10); 14q+ chromosome from Burkittlymphoma (lane 11); chromosome 17 (lane 12); chromosomes 2, 3, 5,8, 12, 17, 20 (lane 13); chromosomes 1, 3-6, 10, 11, 14, 17, 18, X (lane14); chromosomes 1-4, 6, 7, 9, 11, 12, 14, 15, 18, 20-22, X (lane 15);chromosomes 8, 19, 21, 22, X (lane 16); chromosomes 4, 18, X (lane17); chromosomes 4, 6, 12, 20, X (lane 18); chromosomes 4, 6, X (lane19). DNA was digested with an excess of restriction enzyme EcoRI,fractionated in agarose, transferred to nitrocellulose, and hybridizedto nick-translated 32P-labeled phpim5Rl probe. Approximate sizes ofthe mouse- and human-specificpim fragments are shown at left. Eachlane that is positive for the 3.6-kb hpim fragment (lanes 2, 3, 5, 6, 9,14, 15, 18, and 19) represents DNA from a cell line that retains humanchromosome 6; other lanes (negative for hpim) contain DNA fromcells that do not contain human chromosome 6.

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Table 1. Correlation of the presence of the hpim gene andspecific human chromosomes in 17 mouse-human hybrids

Number of hybrid clones

hpim gene/chromosomeHuman retention

chromosome +/+ -/- +/- -/+ Discordant

1 4 9 4 0 42 1 8 7 1 83 4 7 4 2 64 7 6 1 3 45 3 6 5 3 86 8 9 0 0 07 5 7 3 2 58 2 5 6 4 109 3 9 5 0 510 3 9 5 0 511 2 9 6 0 612 2 8 6 1 713 3 8 5 1 614 4 6 4 3 715 1 6 7 3 1016 2 9 6 0 617 5 4 3 5 818 3 6 5 3 819 0 7 8 2 1020 5 6 3 3 621 2 7 6 2 822 3 6 5 3 8X 6 4 2 5 7

A panel of hybrid cells was characterized for the presence ofspecific human chromosomes by isozyme analysis and, in somecases, karyotypic analysis and DNA-DNA hybridization using DNAprobes for genes assigned to specific chromosomes (13-18). DNAfrom these cells was analyzed for the presence of the hpim gene asdescribed in the legend to Fig. 2.

Lower is a graphical representation of the chromosomaldistribution of 160 silver grains on 100 human metaphases; 29grains were found over chromosome region 6pl2-6p21, withmost grains localized at region 6p12. Thus, about 18% of thegrains are located on the proximal halfofchromosome region6p. Taken together, the chromosomal in situ hybridizationand somatic cell hybrid data are consistent with the mappingof the human hpim locus to band 6p21.

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FIG. 3. hpim is on the short arm of chromosome 6. DNA (10 .gper lane) from mouse-human hybrid 56-47 C122, which retainstranslocation chromosome t(6;17)(6pter-6p21::17pl3-17qter) butnot normal 6 or 6p21-6qter (lane 1); mouse-human hybrid 56-47 c122(counterselected with bromodeoxyuridine), which has lost the t(6;17)translocation chromosome (lane 2); and human peripheral bloodlymphocytes was digested with EcoRI and processed as described inthe legend to Fig. 2. The 3.6-kb hpim fragment is present in the hybridretaining the 6p21-pter portion of chromosome 6 (lane 1) but absentin the hybrid that has lost the t(6;17) translocation chromosome.

FIG. 4. hpim maps to human chromosome region 6pl2-6p21.(Upper) Sublocalization of the hpim gene was determined by chro-mosomal in situ hybridization. A G-banded metaphase fromphytohemagglutinin-stimulated lymphocytes of a normal male washybridized with 3H-labeled probe phpim5Rl, as described (16, 18).The arrow indicates a silver grain over the short arm ofchromosome6. (Lower) Histogram showing the grain distribution in 100metaphases. The abscissa represents the banded chromosomes andtheir relative sizes; the ordinate represents the number of silvergrains present on the chromosome bands.

Transcriptional Activity ofthe hpim Gene. To determine thespectrum of tissues in which the hpim gene is expressed, wehybridized cytoplasmic RNA from various cell lines to the32P-labeled phpim5Rl probe. Cytoplasmic RNAs from nineBurkitt lymphoma cell lines; one erythroleukemia (K562);and three lymphoblastoid, one promyelocytic (HL60), onemyeloma, seven T-cell, and two pre-B-cell lines were ana-lyzed for the presence ofhpim transcripts by hybridization ofnick-translated 32P-labeled phpim5Rl probe to denatured,fractionated RNA fixed on nitrocellulose membranes. Thelymphoblastoid cell lines (Fig. 5 A, lane 1, and B, lanes 4 and5), the myeloma line (data not shown), and the Burkitt

1

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Proc. Natl. Acad. Sci. USA 83 (1986) 2559

A B1 2 3 4 5 la 1 2 3 4 5 6

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FIG. 5. Expression of hpim gene in cell lines of hematopoieticorigin. (A) Cytoplasmic RNA (20 ,ug per lane) from lymphoblastoidcell line GM607 (lane 1), T-cell line Jurkat (26) (lane 2), T-cell lineSuptl (27) (lane 3), pre-B-cell line 380 (lane 4), and pre-B-cell line 697(28) (lane 5). (B) Cytoplasmic RNA (20 ,ug per lane) fromerythroleukemia cell line K562 (29) [lanes la (shorter exposure) and1], Burkitt lymphoma cell line BL2 (30) (lane 2), Burkitt lymphomacell line Daudi (lane 3), lymphoblastoid cell line GM1500 (lane 4),lymphoblastoid cell line GM607 (lane 5), and promyelocytic cell lineHL60 (31, 32) (lane 6). Total cytoplasmic RNA was denatured,fractionated in agarose, transferred to nitrocellulose filters, andhybridized to nick-translated 32P-labeled phpim5R1 as described(20). Cell lines with GM numbers are from the Human GeneticMutant Cell Repository, Camden, NJ.

lymphoma cell lines (Fig. 5B, lanes 2 and 3) expressed variouslevels of a 3.2-kb hpim transcript, from barely detectable toreadily detectable. The promyelocytic cell line, HL60, didnot express detectable levels of the 3.2-kb hpim mRNA (Fig.5B, lane 6), nor did most of the T-cell lines tested (forexample, Fig. 5A, lanes 2 and 3); one T-cell line, HSB,expressed readily detectable hpim mRNA of normal length(data not shown). Of the two pre-B-cell lines tested, one, 380(Fig. 5A, lane 4), expressed a low level of hpim mRNA andone, 697, appears to be negative (Fig. 5A, lane 5). Theerythroleukemia cell line K562 expresses a very high level ofhpim mRNA (Fig. 5B, lanes la and 1). This is of interestbecause the K562 cell line contains a marker chromosome,M2, which has been identified as a t(6;6)(pter-pll::p21-qter)translocation chromosome (33). Thus far, we have notdetected rearrangements of the hpim locus in K562 DNA byusing the phpim5Rl probe, but this question will be betterinvestigated when full-length hpim cDNA clones are avail-able. The RNA data thus far demonstrate that the hpim locusis actively transcribed at various levels in many cell lines ofhematopoietic lineage. Further investigation will be requiredto define precisely the tissue spectrum in which the expres-sion of this gene is likely to play a role.

DISCUSSION

The localization to human chromosome region 6p2l of hpim,the human homolog of a murine gene that is activated byretroviral insertion in murine T-cell lymphomas, is of partic-ular interest due to the recent finding of a t(6;9) (p21;q33)translocation chromosome as the sole chromosomal anomalyin several cases of myeloid leukemia (34). In an elegant studyby Vermaelen et al. (34), three patients with myeloprolifera-tive disorders-two acute myeloid leukemias and one chronicmyelocytic leukemia-were found to present with at(6;9)(p21;q33) translocation chromosome which was identi-cal in the three unrelated cases. Vermaelen et al. speculatedthat this translocation, which apparently occurs rarely, mayconstitute a characteristic chromosome anomaly in humanmyeloproliferative disorders. Additionally, there are reportsof t(6;9)(p23;q34) translocations occurring in acute myelo-blastic leukemia (35-38) and of a complex translocation

chromosome, t(6;9;22;11)(p21;q34;qll;ql3) (39) in a patientwith chronic myelocytic leukemia. It is possible that thedifferent interpretation of the breakpoints, 6p21 versus 6p23and 9q33 versus 9q34, reflects a real difference, but given thesensitivity of the present chromosome-banding techniquesand the subtlety of the chromosome anomaly, Vermaelen etal. (34) have concluded that the anomaly is probably the samein the reported cases and that t(6;9) constitutes a character-istic structural chromosome anomaly in acute nonlympho-cytic leukemia (ANLL). When full-length cDNA clones forthe hpim gene are available, it will be possible to determinewhether the hpim gene is directly involved in the t(6;9)translocation of ANLL.

Translocations between 6p2 and 14ql have also beendescribed in human leukemias (40). The location of the achain of the T-cell receptor at 14q11.2 (41) suggests thepossibility that such translocations could result in the acti-vation of the hpim locus by its proximity to the a-chain locusof the T-cell receptor. Inversions and translocations involv-ing region 14q11.2 have been described in T-cell lymphomasand leukemias (27, 42), and the T-cell receptor was found tobe split by chromosome translocations in T-cell malignancies(43). The chromosomal localization ofthe hpim gene suggeststhat this gene may have an important role in human leukemia.Since different translocations, t(6;9) and t(6;14), may result inactivation of the same oncogene, it seems likely that differentactivation mechanisms may be involved in the pathogenesisof these diseases.

We thank Wendy Scattergood and Jim Gorham for expert researchassistance and Dr. Yen Li for the mouse pim-J molecular clone. Thiswork was supported by National Institutes of Health GrantsCA39860, CA10815, CA21124, and HD17561.

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y 20

, 202

0