[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. Abdulkadir1
1
Department of Pathology, 2Division of Hematology and Oncology, and 3High Resolution Imaging Facility and Department of Cell Biology, University of Alabama at
Birmingham School of Medicine, Birmingham, Alabama, and 4Division of Medical Oncology, University of Colorado Health Science Center, Denver, Colorado
Abstract
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 majority 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 function that has been implicated in lymphomagenesis (7). Pim-1 is widely
expressed in tissues, with the highest expression found in hematopoietic 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 identified, the precise roles of Pim-1 in normal cellular physiology or
tumorigenesis remain obscure. Recently, microarray expression profiling 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 prostate epithelial cell lines overexpressing Pim-1. Our analyses indicate
that overexpression of Pim-1 interferes with the mitotic spindle checkpoint, leading to polyploidy and chromosome missegregation.
tagged Mr 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 Biotechnology).
Flow Cytometric Analysis. Cells were fixed with ethanol and stained with
propidium iodide for DNA content analysis. For BrdUrd5 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 following primary antibodies overnight at 4°C: anti-␣-tubulin and anti-␥-tubulin
(Sigma). Secondary antibodies were Alexa Fluor 594 antimouse- or antirabbitFITC 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⬘-CGCCGCTAGAGGTGAAATTCT-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 determined by examination of H&E-stained frozen sections. Total RNA was prepared 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 cytometry.
Materials and Methods
Results
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-
Abnormal Cell Cycle and Polyploidy in Pim-1 Overexpressing
Cells. To evaluate the role of PIM-1 overexpression in prostate carcinogenesis, we established prostate epithelial cell lines that stably
overexpress Pim-1. The immortalized, nontumorigenic prostate epithelial cell line RWPE-1 (12) and the prostate carcinoma cell line
LNCaP were transduced with control retrovirus or retrovirus expressing a FLAG-tagged murine Pim-1 gene. After selection in G418,
Aneuploidy and chromosomal aberrations are hallmarks of most human 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 overexpressing Pim-1 have a defect in the mitotic spindle checkpoint, abnormal
mitotic spindles, centrosome amplification, and chromosome missegregation. 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
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: [email protected].
5
The abbreviations used are: BrdUrd, bromodeoxyuridine; DAPI, 4⬘,6-diamidino-2phenylindole; RT-PCR, reverse transcription-PCR.
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ONCOGENIC KINASE PIM-1 AND GENOMIC INSTABILITY
pools of stable Pim-1 expressing clones were obtained for further
study. Western blot analysis for the FLAG epitope and the Pim-1
protein confirmed protein overexpression in the cell lines (Fig. 1A).
To assess the relative overexpression of Pim-1 in transduced cells, we
used quantitative RT-PCR using primers that recognize both the
human and murine Pim-1 mRNAs. Pim-1 expression levels in RWPEPim-1 and LNCaP-Pim-1 cells were 10.5- and 14.5-fold higher, respectively, than those in control cells (Fig. 1B). We also determined
levels of PIM-1 expression in benign and malignant human prostate
specimens. We identified a tumor with PIM-1 expression that is
⬃30-fold higher than that in normal prostate (Fig. 1B), indicating that
the level of overexpression in our stable cell lines is within the range
seen in human tumors. Initial characterization of the Pim-1-overexpressing cell lines indicate that elevated Pim-1 expression did not
significantly alter the growth rates of the cell lines as determined by
cell counting, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay, or colony forming assay (data not shown).
Recent studies indicating that Pim-1 localizes to the spindle poles
during mitosis and interacts with the nuclear mitotic apparatus protein,
NuMA (13), prompted us to examine whether Pim-1 plays a role in
regulating the cell cycle. Flow cytometric analysis of propidium
iodide-stained, asynchronously growing cells showed a significant
accumulation of cells with 4N DNA content in Pim-1-overexpressing
cells derived from both the nontumorigenic RWPE-1 and the tumorigenic LNCaP cell lines (Fig. 1, C and D). Furthermore, Pim-1
overexpression led to polyploidy (DNA content ⬎4N). Notably,
polyploidy in Pim-1-overexpressing cells occurred without induction
of microtubule stress, but can be additionally increased by treating the
cells with the antimicrotubule agent Taxol (Fig. 1, C–E). The
polyploidy observed in Pim-1-overexpressing cells suggests that cells
with a 4N DNA content are able to undergo an additional round of
DNA replication without prior cell division, resulting in cells with 8N
DNA content. To demonstrate this directly, we performed BrdUrd
incorporation experiments. In RWPE-Neo cells, BrdUrd incorporation
Fig. 1. Tetraploidy and polyploidy after Pim-1 overexpression. A, Western blot analysis of Pim-1 expression in RWPE-1 and LNCaP human prostate cell lines expressing
FLAG-tagged murine Pim-1 or empty vector (Neo). Blots were probed with antibodies against Pim-1, the FLAG epitope, and actin. B, quantitative RT-PCR analysis of Pim-1 expression
in prostate cell lines and dissected benign (P9 and P10) and malignant (P3 and P8) human prostate specimens. Pim-1 levels were assessed using primers that amplify both human and
mouse Pim-1, and 18S rRNA was used for normalization. The means from triplicate measurements are shown; bars, ⫾SD. C and D, Cell cycle profiles of RWPE-1 and LNCaP cells
(with or without Taxol treatment) as determined by flow cytometry after propidium iodide staining. E, percentage of cells with DNA content ⬎4N after Taxol treatment. F,
two-dimensional analysis of BrdUrd incorporation (Y-axis) and DNA content (X-axis) by flow cytometry. Note BrdUrd incorporation by cells with DNA content between 4N and 8N.
All of the experiments were replicated two to six times.
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ONCOGENIC KINASE PIM-1 AND GENOMIC INSTABILITY
Fig. 2. Abnormal microtubule organization and genomic instability in Pim-1-overexpressing cells. A and B, mitotic index of unsynchronized RWPE-Neo and RWPE-Pim-1 cells
treated with 10 nM Taxol and stained for MPM-2 (A) or phosphohistone H3 (B) to determine the mitotic index by flow cytometry. C–F, control Neo and Pim-1 expressing RWPE-1
and LNCaP cells were stained with ␣-tubulin antibody (red) and DAPI (blue) to mark microtubules. Multinucleated cells are marked with arrows. G, percentage of cells with multiple
nuclei. At least 300 cells were counted for each cell line from two independent experiments. H, percentage of cells containing micronuclei obtained by counting at least 500 cells from
two independent experiments.
is present mostly in the S phase fraction between 2N and 4N (Fig. 1F).
By contrast, in RWPE-Pim-1 cells the most active BrdUrd incorporation was seen in the population of cells with DNA content between
4N and 8N (Fig. 1F), confirming that the 8N cells arose by rereplication of tetraploid cells. Thus, elevated expression of Pim-1 leads to
polyploidy by subverting the checkpoints that prevent DNA rereplication without prior cell division.
Mitotic Checkpoint Defect in Pim-1-Overexpressing Cells. The
integrity of the mitotic spindle checkpoint in Pim-1-overexpressing
cells was assessed by monitoring the mitotic index in cells treated
with Taxol for various time points. The mitotic index was determined
by staining cells with MPM-2 antibody, which recognizes mitosisspecific phosphoepitopes (14). Control RWPE-Neo cells showed a
robust mitotic arrest, with 40% of the cells arrested by the 18 h time
point, compared with only 8% of RWPE-Pim-1 cells (Fig. 2A). We
also monitored phosphorylation of histone H3 as an additional mitotic
marker. Histone H3 phosphorylation begins on entry into mitosis, and
appears to be important for chromosome condensation and segregation (15). After 24-h Taxol treatment, 52% of RWPE-Neo cells were
phospohistone H3 positive, compared with 18% of RWPE-Pim-1 cells
(Fig. 2B). A similar mitotic checkpoint defect was observed in LNCaP
cells, where 30% of LNCaP-Pim-1 cells were phosphohistone H3
positive after 18 h Taxol treatment compared with 9% of LNCaP-Neo
cells (data not shown). In sum, these results indicate that Pim-1overexpressing cells have a mitotic checkpoint defect.
Spindle Defects and Chromosome Missegregation in Pim-1
Overexpressing Cells. Loss of the mitotic spindle checkpoint may
lead to chromosomal instability and polyploidy by affecting chromo-
some segregation. Therefore, we examined the microtubule network,
mitotic spindle, and centrosomes in Pim-1-overexpressing cells. Cells
were stained with ␣-tubulin antibody to highlight the microtubules.
Control RWPE-Neo and LNCaP-Neo cells presented a flattened morphology, with dendritic extensions and a fine network of cytoplasmic
microtubules (Fig. 2, C and E). By contrast, the Pim-1-overexpressing
cells (particularly RWPE-Pim-1 cells) were more rounded-up with
uneven microtubule staining (Fig. 2, D and F). An increase in the
number of multinucleated cells was also apparent in both RWPEPim-1 and LNCaP-Pim-1 cells (Fig. 2G) consistent with our flow
cytometry data showing polyploidy in Pim-1-overexpressing cells
(Fig. 1). Another notable feature of Pim-1-overexpressing cells is an
increase in the number of micronuclei (Fig. 2H). Micronuclei arise by
chromosome missegregation during mitosis and appear in the cytoplasm as DNA-containing spheres surrounded by a nuclear membrane. The extent of micronucleus formation is an indication of the
frequency of cells in a population that is losing chromosomes and is
a marker of aneuploidy (16).
We next examined the status of the mitotic spindles and centrosomes in Pim-1-overexpressing cells. Cells were costained with antibodies to ␣-tubulin (to mark spindles) and ␥-tubulin (to mark centrosomes). Examination of mitotic cells revealed spindle abnormalities in
a majority of Pim-1-overexpressing cells. Whereas most RWPE-Neo
cells in mitosis establish strong bipolar spindles with normal chromosomal congression (Fig. 3A), a significant fraction of RWPE-Pim-1
cells in mitosis showed a range of abnormalities (Fig. 3, B–D). These
abnormalities include disorganized, weakly stained, or multipolar
spindles, and misaligned or missegregated chromosomes. These ab-
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ONCOGENIC KINASE PIM-1 AND GENOMIC INSTABILITY
Fig. 3. Centrosomal and spindle abnormalities in Pim-1-overexpressing cells. Cells were stained to mark spindles (␣-tubulin, red), centrosomes (␥-tubulin, green), and DNA (DAPI,
blue). Arrows indicate unaligned chromosomes. A, dontrol RWPE-Neo cell showing normal bipolar spindle. B–D, examples of abnormalities seen in RWPE-Pim-1 cells, including
disorganized, weakly stained spindles, lagging and misaligned chromosomes (arrows, B and C), and multipolar spindles (D).
normalities were seen in 53% of RWPE-Pim-1 cells compared with
21% of RWPE-Neo cells (⬎200 mitoses counted from three independent experiments).
An increase in the number of cells with multiple (⬎2) centrosomes
was also apparent in Pim-1-overexpressing cells compared with controls (15.7% versus 5.8% of ⬎300 cells counted from three experiments). Centrosome amplification may result from defective cell
division and multinucleation, leading to amplification of centrosome
number during subsequent cell cycles (5, 17). Alternatively, direct
dysregulation of the centrosome duplication cycle can lead to centrosome amplification (5, 17). We favor the former possibility in Pim1-overexpressing cells, because these cells have a checkpoint defect
that leads to polyploidization and multinucleation.
Delayed Cytokinesis in Pim-1-Overexpressing Cells. The existence of a cytokinesis checkpoint was described recently in yeast (18).
In a cell with a misaligned spindle in which the cell fails to correct the
defect, polyploidy ensues because of a failure to complete cytokinesis
(18). The results presented thus far indicating abnormal spindle assembly, loss of checkpoint control, polyploidy, and multinucleation in
Pim-1-overexpressing cells all suggest that these cells have a cytokinesis delay. To observe this directly, we used time-lapse video microscopy. We followed cells by both differential interference contrast
(DIC) and fluorescence microscopy after staining the DNA with
Hoechst 33258 dye. Single cells about to enter mitosis were photographed every 15 min (Fig. 4). Under the experimental conditions we
used, a control cell completed cell division in 180 min (Fig. 4). The
Pim-1-overexpressing cell, on the other hand, is still in anaphase at
this time, and took an additional 180 min to complete division (Fig. 4).
Pim-1-overexpressing cells also had less compacted chromosomes
and lagging chromosomes (Fig. 4, arrows).
To demonstrate cytokinesis delay at a cell population level, we
followed the cell division rate using the fluorescent cell linker PKH67
dye. The decrease in fluorescent intensity as the dye is dispersed
between the daughter cells during cell division has been used for cell
division tracking (6). It took 1.5 days to achieve 50% decrease of
median fluorescent dye intensity in RWPE-Neo cells, whereas this
took 3 days in RWPE-1-Pim-1 cells (n ⫽ 2). Together these data
indicate that Pim-1-overexpressing cells have defective cytokinesis.
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ONCOGENIC KINASE PIM-1 AND GENOMIC INSTABILITY
Fig. 4. Delayed cytokinesis and chromosome lag in Pim-1-overexpressing cells. RWPE-Neo (Neo) and RWPE-Pim-1 (Pim-1) cells were stained with Hoechst 33258 and followed
through mitosis by time-lapse videomicroscopy, and DIC and fluorescent images were captured every 15 min. Only some are shown, with time points in minutes indicated. Pim-1 cells
show a delay in cytokinesis and chromosome lag (arrows in the 300-min time point).
Discussion
Chromosome alignment and segregation during mitosis are critically dependent on the normal formation of a bipolar mitotic spindle.
In the present work, we have identified a novel role for Pim-1overexpression in overriding the mitotic spindle checkpoint, resulting
in genomic instability. Pim-1 was shown recently to interact with and
phosphorylate the nuclear mitotic apparatus protein, NuMA (13). It
was proposed that Pim-1 is important for maintaining a stable complex linking the chromosomal kinetochores to the spindle microtubules consisting of NuMA, dynein/dynactin, and HP1␤ (13). Our
results suggest that overexpression of Pim-1 could affect spindle
dynamics by disrupting the normal function of this complex.
Due to loss of checkpoint control, Pim-1-overexpressing cells with
spindle abnormalities are not arrested in mitosis, but they fail to
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ONCOGENIC KINASE PIM-1 AND GENOMIC INSTABILITY
efficiently complete cytokinesis. This results in polyploidy and
multinucleation. Therefore, Pim-1 overexpression represents a novel
mechanism that can be used by tumors to override the mitotic spindle
checkpoint. At present, the mechanism of Pim-1 subversion of the
mitotic checkpoint is not clear; however, we have observed that
Pim-1-induced polyploidy is passage dependent, implying the need
for accumulation of additional changes.6
Expression studies of PIM-1 in human prostate cancer present a
paradox in that although PIM-1 expression is increased in a majority
of prostate carcinomas compared with benign prostate tissue, poor
clinical outcome is correlated with decreased expression of PIM-1
(10). Our results linking PIM-1 expression to genomic instability may
help explain this paradox. Early in the process of tumorigenesis,
genomic instability is thought to promote the acquisition of protumorigenic mutations, which are selected for during the course of
tumor progression. However, when a tumor has acquired an abnormal
genome that confers growth advantage, it is advantageous for these
genetic changes to be “fixed.” This has been referred to as genetic
convergence (19). Thus, PIM-1 overexpression may be important for
driving genomic instability in early tumors, whereas advanced tumors
down-regulate PIM-1 to stabilize any acquired abnormalities.
In summary, our results identify a novel role for Pim-1 overexpression in inducing genomic instability through effects on the mitotic
spindle checkpoint. These findings generate several hypotheses regarding the value of Pim-1 as a biomarker and as a therapeutic target
in prostate tumors.
Acknowledgments
We thank Scott Ness and Gerard Evan for the generous gift of reagents, and
Torey Combs, Andra Frost, Cynthia Moore, Enid Keyser, and Tracey McGuire
for technical assistance.
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Overexpression of the Oncogenic Kinase Pim-1 Leads to
Genomic Instability
Meejeon Roh, Bernard Gary, Chisu Song, et al.
Cancer Res 2003;63:8079-8084.
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