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Human Mortalin (HSPA9): a Candidate for the Myeloid Leukemia Tumor Suppressor Gene on 5Q31 H Xie1,Zhu1, B Chyna1, SK Horrigan2 and CA Westbrook1

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Human Mortalin (HSPA9): a Candidate for the Myeloid Leukemia Tumor Suppressor Gene on 5Q31 H Xie1,Zhu1, B Chyna1, SK Horrigan2 and CA Westbrook1 Leukemia (2000) 14, 2128–2134 2000 Macmillan Publishers Ltd All rights reserved 0887-6924/00 $15.00 www.nature.com/leu Human mortalin (HSPA9): a candidate for the myeloid leukemia tumor suppressor gene on 5q31 H Xie1,ZHu1, B Chyna1, SK Horrigan2 and CA Westbrook1 1Section of Hematology/Oncology, Department of Medicine, University of Illinois at Chicago, Chicago, IL; and 2Department of Pediatrics, Georgetown University, Washington DC, USA Human mortalin (HSPA9) was originally identified by its close cDNAs for both the pancytosolic form (mot-1) and the peri- homology to murine mortalins, which play important roles in nuclear form (mot-2) of murine mortalin have been fully cellular senescence. The two murine genes, mot-1 and mot-2, cloned, and the genomic organization of mot-2 has been differ in only two amino acid residues, but have opposite func- 1,3 tions in cellular immortalization. HSPA9 was recently localized reported. Recent evidence suggests that the two mortalins to chromosome 5, band q31, a region that is frequently deleted are not isoforms, but instead originate from two distinct gen- in myeloid leukemias and myelodysplasia (MDS), making it a omic loci. Fluorescence in situ hybridization assigned the candidate tumor suppressor gene, which is consistent with the mortalin-related genes to mouse chromosomes 18 and X.6 The biological function of its murine homologue. To evaluate mor- two forms of mortalin are variably present in different strains talin in this capacity, its expression in normal and leukemic cell lines was investigated, and its genomic structure was deter- of mouse; thus, both mot-1 and mot-2 are present in the mined in order to facilitate mutation detection. RT-PCR and Balb/c strain, while Swiss and C56BL/6 mice contain only Northern blot analysis revealed a broad distribution in normal mot-2, and C3H He mice contain only mot-1. It has been tissues and in leukemia cell lines, producing a single 2.8 kb suggested that this distribution of mortalin genes may explain transcript. Genomic characterization showed that the gene the different rates of spontaneous immortalization of cells spans 18 kb, and consisted of 17 exons with boundaries that found in different strains of mouse.5 were almost identical to its murine counterpart. Using intron- based primers to flank each exon, sequence of the complete Unlike murine mortalin, there appears to be only one protein-coding regions was obtained for three AML cell lines, human mortalin gene, HSPA9, which is highly homologous including two lines with chromosome 5 loss (KG-1 and HL-60) to both mot-1 and mot-2, differing from them in amino acid and one without (AML-193) compared to normal DNA. No sequence by only 6 and 5 residues, respectively. HSPA9 has mutations were identified although one conservative nucleo- been assigned to chromosome 5 band q31.1.6 Recently, we tide sequence variant was observed in exon 16. We have shown refined the location of this gene to a 700-kb interval, which that mortalin is highly conserved in genomic structure as well we postulate to be the site of a tumor suppressor gene impli- as sequence, and the designed primers will be suitable for − future studies to detect mutations in clinical samples. Leukemia cated in myeloid malignancies manifesting 5 or del(5q), (2000) 14, 2128–2134. including acute myeloid leukemia (AML) and myelodysplasia Keywords: mortalin; HSPA9; genomic structure; mutation; tumor (MDS).7 The chromosomal location of the mortalin gene, its suppressor biologic function in murine cells, and its association with cellular immortalization, make it an appealing candidate for a tumor suppressor gene whose mutational loss or inactivation Introduction results in transformation of myeloid cells. Here, we report the genomic structure of human mortalin, which we found to be Human mortalin (also called peptide-binding protein 74 highly similar in organization to murine mot-2. Additionally, (PBP74), and heat shock 70kD protein 9 (HSPA9)) is an inter- we report intron-based primers, suitable for genomic sequen- esting gene that shows a high degree of nucleotide sequence cing, and their use to evaluate the mortalin coding sequence homology to its murine counterparts mortalin-1 (mot-1) and in several myeloid leukemia cell lines with −5 or del(5q). mortalin-2 (mot-2). The mouse homologues were initially identified by their roles in cellular senescence, where they appear to have contrasting functions. In non-immortalized murine fibroblasts, mortalin displays a uniform pancytosolic distribution, whereas it manifests a perinuclear location in Materials and methods immortalized fibroblasts.1,2 Cytosolic mot-1 has been pro- posed to be a major determinant of fibroblast senescence, such that its transfection into NIH3T3 cells induces cellular Cell culture and bacterial clones senescence, whereas microinjection of anti-mortalin antibody can stimulate proliferation in senescent murine fibroblasts.3 On the other hand, over-expression of mot-2 can induce Cell lines used in this study were obtained from ATCC malignant transformation of NIH 3T3 cells.4 The cytosolic and (Rockville, MD, USA). Cells were cultured using rec- perinuclear forms of mortalin were later shown to arise from ommended conditions, or in RPMI-1640 medium (GIBCO the two related genes, mot-1 and mot-2, which differ from BRL, Grand Island, NY, USA) supplemented with 10% heat- each other by only two amino acid residues, respectively.5 inactivated fetal calf serum (Sigma, St Louis, MO, USA) at ° + 37 C in an atmosphere of 5% CO2 in air. CD34 cells were isolated from bone marrow obtained from normal donors with informed consent under the guidelines provided by the Uni- Correspondence: CA Westbrook, University of Illinois at Chicago, + Departments of Medicine and Genetics, M/C 734, 900 S Ashland, versity of Illinois. The CD34 cells were isolated and purified 8 Chicago, Illinois 60607–7170, USA; Fax: 312–413–7963 as previously described. BAC clone 15L17 was obtained from Received 22 May 2000; accepted 1 August 2000 Research Genetics (Huntsville, AL, USA). Mortalin as a candidate for tumor suppressor gene on 5q31 H Xie et al 2129 Preparation of DNA and RNA hybridized with a ␤-actin probe as an internal control of both the RNA integrity and amount. A multiple tissue Northern Genomic DNA and total cellular RNA were extracted using blot, MTN H, (Clontech, Palo Alto, CA, USA), which con- TRIZol Reagent (Gibco Technologies, Gaithersburg, MD, tained mRNAs from various human tissues including heart, USA) according to the recommended protocol from 107 brain, placenta, lung, liver, skeletal muscle, kidney and pan- CD34+ cells, or from cell lines that were harvested in the log- creas was used to detect expression of mortalin. A hybridiz- arithmic growth phase. BAC DNA was prepared according to ation probe was prepared from a 649-bp fragment of human standard protocols used in this laboratory.9 mortalin containing sequences from exon 9 to exon 11 and amplified by RT-PCR from human leukemia cell line KG-1. The pair of primers used consisted of the forward primer for Reverse-transcriptase PCR (RT-PCR) exon 9 (5Ј-CTGCTGAAAAGGCTAAATGTGAACTCTC-3Ј) and the reverse primer for exon 11 (5Ј-CACAGCCTCATCAG- RT-PCR was performed using either the Titan One Tube RT- GATTGAGAGCTT-3Ј). The RT-PCR product was labelled with PCR System (Boehringer Mannheim, Indianapolis, IN, USA) ␣-32P-dCTP by random primer labelling using Multiprime or Enhanced Avian RT-PCR kit (Sigma, St Louis, MO, USA) DNA Labeling System(Amersham Pharmacia Biotech, Piscata- according to the manufacturer’s protocol. Optimal Mg2+ con- way, NJ, USA). centrations and annealing temperatures were determined for each primer pair. All reactions were performed in duplicate, including control reactions which did not include reverse transcriptase or RNA. PCR reactions were performed in a Per- kin-Elmer (Foster City, CA, USA) 480 Thermocycler using the Results following parameters: initial denaturing at 94°C for 3 min, fol- lowed by 30 cycles of denaturation at 94°C for 40 s, annealing at 52–60°C, (optimized for each primer pair) for 1 min, and Genomic organization of human mortalin extension at 72°C for 1 min, with a final extension at 72°C for 7 min. Each 20 ␮␭ of PCR reaction contains 100 ng of template DNA, 10 mM Tris HCl, pH 8.0, 2.0 mM MgCl, 200 The intron–exon boundaries of human mortalin were pre- ␮M each of dATP, dTTP, dCTP and dGTP, 15 pmol of each dicted by alignment of the human cDNA sequence (GenBank primer and 0.5 units of Taq DNA polymerase (Bioline, IL, Accession No. L15189 ) with the published intron–exon USA). PCR products were separated by agarose gel electro- boundary sequences of murine mot-2 gene10 using Seqman phoresis and visualized with ethidium bromide, using a 100 software. Exon-based primers at the presumed intron–exon bp or 1 kb DNA ladder (GIBCO BRL) for size estimation. boundaries were then designed to amplify intron sequences Long-range PCR was performed using the Expand 20kbPlus between the exons (Table 1) using DNA from BAC clone PCR System (Boehringer Mannheim, Indianapolis, IN, USA) 15L17 as a template. This BAC clone was identified as con- according to the manufacturer’s instructions. taining the genomic sequence for mortalin by BAC typing in our laboratory (data not shown). In those instances in which the predicted PCR product appeared to be longer than 3.0 kb DNA sequencing and failed to amplify, such as intron 9 (5.0 kb), long-range PCR was attempted using the Expand 20 kbPlus PCR System The DNA sequencing reaction was performed using the ABI according to the manufacturer’s instruction (Boehringer Prism BigDye Terminator Sequencing kit (Perkin-Elmer). In Mannheim). The PCR products obtained from this exon-to- brief, PCR products amplified from each exon were purified exon amplification were separated by agarose gel electro- using Qiagen PCR purification kit (Qiagen, Valencia, CA, phoresis and the size of each intron was estimated by compar- USA).
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