[CANCER RESEARCH 63, 8079 – 8084, December 1, 2003] Advances in Brief Overexpression of the Oncogenic Kinase Pim-1 Leads to Genomic Instability Meejeon Roh, 1 Bernard Gary, 1 Chisu Song, 2 Nasser Said-Al-Naief, 1 Albert Tousson, 3 Andrew Kraft, 4 Isam-Eldin Eltoum, 1 and Sarki A. Abdulkadir 1 1 Department of Pathology, 2 Division of Hematology and Oncology, and 3 High Resolution Imaging Facility and Department of Cell Biology, University of Alabama at Birmingham School of Medicine, Birmingham, Alabama, and 4 Division of Medical Oncology, University of Colorado Health Science Center, Denver, Colorado Abstract Aneuploidy and chromosomal aberrations are hallmarks of most hu- man epithelial malignancies. Here we show that overexpression of the oncogenic kinase Pim-1 in human prostate epithelial cells induces genomic instability by subverting the mitotic spindle checkpoint. Cells overexpress- ing Pim-1 have a defect in the mitotic spindle checkpoint, abnormal mitotic spindles, centrosome amplification, and chromosome missegrega- tion. Polyploidy and aneuploidy ensue due to a delay in completing cytokinesis. These results define a novel role for elevated Pim-1 expression in promoting genomic instability in human prostate tumors. Introduction Faithful segregation of the genome during cell division in human cells is dependent on the normal function of the mitotic spindle. Defects in the organization, constitution, or regulation of the mitotic spindle apparatus are thought to be important causes of chromosome missegregation and aneuploidy in human cancer (1). The molecular mechanisms underlying the development of aneuploidy in the major- ity of human cancers are not fully defined, although mutations in mitotic checkpoint genes such as hBUB1 and CHFR have been identified in a subset of human cancers and cell lines (2, 3). The development of aneuploidy has also been associated with mutations/ overexpression of several tumor suppressor genes and oncogenes, including BRCA1/2, p53, pRb, Myc, Ras, Mos, and AURORA-A (4 – 6). Pim-1 is an oncogenic serine-threonine kinase of ill-defined func- tion that has been implicated in lymphomagenesis (7). Pim-1 is widely expressed in tissues, with the highest expression found in hematopoi- etic tissues and testes (7), and can be induced by a number of cytokines in hematopoietic cells where it enhances cellular survival (8, 9). Although several substrates of Pim-1 kinase have been identi- fied, the precise roles of Pim-1 in normal cellular physiology or tumorigenesis remain obscure. Recently, microarray expression pro- filing identified PIM-1 overexpression in a significant proportion of human prostate tumors (10). To explore the roles of Pim-1 in prostate carcinogenesis, we established nontumorigenic and tumorigenic pros- tate epithelial cell lines overexpressing Pim-1. Our analyses indicate that overexpression of Pim-1 interferes with the mitotic spindle check- point, leading to polyploidy and chromosome missegregation. Materials and Methods Cell Lines and Retroviral Constructs. RWPE-1 and LNCaP cell lines were obtained from American Type Culture Collection and maintained as recommended. MSCV-Pim-1 retrovirus vector was made by inserting FLAG- tagged M r 33,000 murine Pim-1 cDNA into the MSCVneoEB. Virions were produced in ecotropic Phoenix cells and used to infect cell lines as described (9). After selection in 200 –500 g/ml G418, the resistant clones were pooled. Western Blot Analysis. Total cell lysates were resolved by SDS-PAGE and processed for Western blot analysis. Antibodies used are FLAG M2 (Sigma), Pim-1 (Santa Cruz Biotechnology), and actin (Santa Cruz Biotech- nology). Flow Cytometric Analysis. Cells were fixed with ethanol and stained with propidium iodide for DNA content analysis. For BrdUrd 5 analysis, cells were pulsed with 10 M BrdUrd (Sigma) for 30 min before harvesting, then stained with anti-BrdUrd-FITC-conjugated antibody (Becton Dickinson). For MPM2 and phosphohistone H3 staining, the cells were incubated with either MPM2 or antiphophohistone H3 antibodies (Upstate) for 30 min, washed in PBS, and then stained with antimouse or antirabbit FITC-conjugated antibodies (Sigma). Immunofluorescence. Cells on cover slides were incubated with the fol- lowing primary antibodies overnight at 4°C: anti--tubulin and anti--tubulin (Sigma). Secondary antibodies were Alexa Fluor 594 antimouse- or antirabbit- FITC conjugated IgG (Molecular Probes). After counterstaining with DAPI (Sigma), images were captured with a Zeiss Axioskop 40 microscope. Quantitative RT-PCR Analysis. RNA isolation and quantitative RT-PCR (TaqMan; Applied Biosystems) using SYBR-GREEN dye were performed as described (11). PCR reactions were performed in triplicate. Primers used are: Pim-1 Forward, 5'-CGAGTGCCCATGGAAGTGGT-3'; Pim-1 Reverse, 5'- CGGGCCTCTCGAACCAGT-3'; 18S Forward, 5'-CGCCGCTAGAGGT- GAAATTCT-3'; and 18S Reverse, 5'-CGAACCTCCGACTTTCGTTCT-3'. Human Prostate Tumor Specimens. Radical prostatectomy specimens were obtained from the University of Alabama Tissue Procurement Center. Glandular tissue was grossly dissected from samples with no evidence of carcinoma (benign) or samples with 20 –90% carcinoma (malignant) as deter- mined by examination of H&E-stained frozen sections. Total RNA was pre- pared from the tissue using TRIzol (Life Technologies, Inc.). Due to the infiltrative nature of prostate cancer, the determined PIM-1 levels in these samples should be considered a minimum estimate. Time-Lapse Imaging and Analysis. Cells were plated on bottom glass dishes (Warner Instrument). The cells were stained with Hoechst 33258 (Sigma) and maintained at 37°C. Differential interference contrast (DIC) and fluorescence images were captured every 15 min using a 40 objective on a Zeiss Axiovert 200 M microscope. Cell Division Tracking. Cells were labeled with the dye PHK67 (Sigma) following the manufacturer’s instruction. After labeling, cells were replated and harvested every 2 days, and PKH intensity was measured by flow cy- tometry. Results Abnormal Cell Cycle and Polyploidy in Pim-1 Overexpressing Cells. To evaluate the role of PIM-1 overexpression in prostate car- cinogenesis, we established prostate epithelial cell lines that stably overexpress Pim-1. The immortalized, nontumorigenic prostate epi- thelial cell line RWPE-1 (12) and the prostate carcinoma cell line LNCaP were transduced with control retrovirus or retrovirus express- ing a FLAG-tagged murine Pim-1 gene. After selection in G418, Received 6/13/03; revised 9/16/03; accepted 10/8/03. Grant support: NIH (CA94858) and the Howard Hughes Medical Institute (S. A. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Sarki A. Abdulkadir, Department of Pathology, 533 LHRB, University of Alabama at Birmingham School of Medicine, Birmingham, AL 35294-0007. Phone: (205) 975-0730; Fax: (205) 975-9927; E-mail: sabdulka@path.uab.edu. 5 The abbreviations used are: BrdUrd, bromodeoxyuridine; DAPI, 4',6-diamidino-2- phenylindole; RT-PCR, reverse transcription-PCR. 8079 Research. on May 27, 2016. © 2003 American Association for Cancer cancerres.aacrjournals.org Downloaded from