Aberrant expression of serine/threonine kinase Pim-3 in hepatocellular
carcinoma development and its role in the proliferation of human hepatoma
Fujii, Chifumi; Nakamoto, Yasunari; Lu, Peirong; Tsuneyama, Koichi;
Popivanova, Boryana K.; Kaneko, Shuichi; Mukaida, Naofumi
CitationInternational Journal of Cancer, 114: 209-218
Title: Aberrant expression of serine/threonine kinase Pim-3 in hepatocellular
carcinoma development and its role in the proliferation of human hepatoma cell
Chifumi Fujii1, 2, Yasunari Nakamoto3, Peirong Lu1, Koichi Tsuneyama4, Boryana K.
Popivanova1, Shuichi Kaneko3, and Naofumi Mukaida1, 2, *
1Division of Molecular Bioregulation and 2Center for the Development of Molecular
Target Drugs, Cancer Research Institute, 3Department of Gastroenterology, Graduate
School of Medical Science, Kanazawa University, 13-1 Takara-machi, Kanazawa,
Ishikawa 920-0934, and 4Department of Surgical Pathology, Toyama Medical and
Pharmaceutical University Hospital, 2630 Sugitani, Toyama 930-0194, Japan
Short running title: Pim-3 in hepatocellular carcinoma
*Correspondence to Division of Molecular Bioregulation, Cancer Research Institute,
Kanazawa University, 13-1 Takara-machi, Kanazawa, Ishikawa 920-0934, Japan.
Tel: +81-76-265-2767; Fax: +81-76-234-4520;
Key Words: protein serine-threonine kinases, pre-malignant lesions, hepatocellular
carcinoma, RNA interference, apoptosis
The abbreviations used are: BSA, bovine serum albumin; DMEM, Dulbecco’s modified
Eagle’s medium; FBS, fetal bovine serum; FDD, fluorescent differential display;
GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HBV, hepatitis B virus; HCV,
hepatitis C virus; HBs, HBV surface; HBsAg, HBV surface antigen; HBsTg, HBs
transgenic mice; HCC, hepatocellular carcinoma; IL, interleukin; PBS (-), phosphate
buffered saline; PCR, polymerase chain reaction; RNAi, RNA interference; RT-PCR,
reverse transcription-polymerase chain reaction; siRNA, short interfering RNA; STAT,
signal transducers and activators of transcription; VCP, valosine containing protein.
DNA Data Bank of Japan Accession Number AB114795
Journal Category: Cancer Cell Biology
Most cases of human hepatocellular carcinoma develop after persistent chronic
infection with human hepatitis B virus or hepatitis C virus, and host responses are
presumed to have major roles in this process. To recapitulate this process, we have
developed the mouse model of hepatocellular carcinoma using hepatitis B virus surface
antigen transgenic mice. In order to identify the genes associated with
hepatocarcinogenesis in this model, we compared the gene expression patterns between
pre-malignant lesions surrounded by hepatocellular carcinoma tissues and control liver
tissues by using a fluorescent differential display analysis. Among the genes which
were expressed differentially in the pre-malignant lesions, we focused on Pim-3, a
member of a proto-oncogene Pim family, because its contribution to
hepatocarcinogenesis remains unknown. Moreover, the unavailability of the
nucleotide sequence of full-length human Pim-3 cDNA prompted us to clone it from the
cDNA library constructed from a human hepatoma cell line, HepG2. The obtained
2,392 bp human Pim-3 cDNA encodes a predicted open reading frame consisting of 326
amino acids. Pim-3 mRNA was selectively expressed in human hepatoma cell lines,
but not in normal liver tissues. Moreover, Pim-3 protein was detected in human
hepatocellular carcinoma tissues and cell lines but not in normal hepatocytes.
Furthermore, cell proliferation was attenuated and apoptosis was enhanced in human
hepatoma cell lines by the ablation of Pim-3 gene with RNA interference. These
observations suggest that aberrantly expressed Pim-3 can cause autonomous cell
proliferation and/or prevent apoptosis in hepatoma cell lines.
Hepatocellular carcinoma (HCC) ranks the eighth cause of death among human
cancers and is endemic in Asia, Africa, and southern Europe. Most cases of HCC arise
from persistent chronic infection with human hepatitis B virus (HBV) or hepatitis C
virus (HCV)1. Host responses are presumed to be involved in the development of
HCC among patients harboring HBV or HCV, because these viruses lack apparent
oncogenes and the infected patients develop HCC after suffering from chronic
hepatitis-related pathology2, 3.
Hepatitis virus infection induces the generation of virus antigen-specific
cytotoxic T lymphocytes, which have been implicated in both the eradication of viruses
and liver injury4. Cycles of cytotoxic T lymphocyte-mediated liver cell destruction
and regeneration are thought to prepare the mitogenic environment4, 5. In order to
elucidate the molecular and cellular mechanism of HCC initiation and development, one
of us (Y. Nakamoto) has established a mouse model of HCC by using HBV surface
antigen (HBsAg) transgenic mice (HBsTg)6. In this model, bone marrow cells and
splenocytes were obtained from syngeneic wild-type mice, which were immunized with
HBsAg and were transplanted into HBsTg mice, which were myeloablated beforehand.
At 15 months after the transplantation, the transgenic mice developed multiple foci of
HCC surrounded by non-malignant areas consisting of hepatocytes with atypical
nuclear configuration6, 7.
To obtain the molecular insights on hepatocarcinogenesis, we compared the
gene expression pattern between non-tumor portion of this model as a pre-malignant
lesion and normal tissues by using a fluorescent differential display (FDD) analysis.
We observed that several genes are expressed differentially in this pre-malignant lesion,
compared with normal liver tissues. Of interest is that the gene expression of Pim-3,
originally identified as depolarization-induced gene in a rat pheochromocytoma cell
line8, was enhanced in this pre-malignant lesion. Here, we demonstrated that Pim-3
was expressed aberrantly in human HCC tissues and hepatoma cell lines but not normal
liver tissues. We also provided evidence to suggest the involvement of Pim-3 in the
proliferation of human hepatoma cell lines.
MATERIALS AND MATHODS
HBsAg transgenic mouse lineage 107-5D (official designation
Tg[Alb-1,HBV]Bri66; inbred B10D2, H-2d) was provided by Dr. F. V. Chisari (The
Scripps Research Institute, La Jolla, CA)9. Lineage 107-5D contains the entire HBV
envelope-coding region (subtype ayw) under the transcriptional control of the mouse
albumin promoter, and expresses the HBV small, middle, and large envelope proteins in
their hepatocytes9. They display no evidence of liver disease during their lifetime
unless they receive the adoptive transfer of HBsAg-specific cytotoxic T lymphocytes9,
due to their immunological tolerance to the HBs transgene at the T cell level10.
Chronic hepatitis-related liver disease model was generated as described
previously6. Briefly, after male HBsAg transgenic mice were thymectomized and
irradiated (900 cGy), their hematopoietic system was reconstituted with the bone
marrow cells from syngeneic non-transgenic B10D2 (H-2d) mice. At 1 week after the
bone marrow transplantation, the animals received 108 splenocytes from syngeneic
non-transgenic B10D2 (H-2d) mice that were infected intraperitoneally with a
recombinant vaccinia virus expressing HBsAg 3 wk before the splenocyte transfer. At
12 to 15 months after the lymphocyte transfer, multiple HCC foci developed in mice6, 7.
Non-tumor and tumor portions were demarcated macroscopically and were removed
separately. A pathologist without a prior knowledge on the experimental procedures
confirmed the presence of hepatocytes with atypical configurations but not malignant
cells in this non-tumor potion. Hence, non-tumor portions were designated as
pre-malignant lesions in the following experiments. Liver tissues were also obtained
from untreated or HBsTg mice transplanted with tolerant splenocytes as a control.
Fluorescent differential display
Total RNAs extracted from liver tissues were subjected to FDD according to
the method described by Ito and colleagues11. Briefly, total RNAs were isolated with
RNA-Bee (Biotecx Laboratories, Inc.), followed by the treatment with RNase-free
DNase (Takara Shuzo, Kyoto, Japan). The purified total RNAs (2.5 µg) were
reverse-transcribed with SuperScript II reverse transcriptase (Invitrogen) and
fluorescein-labeled anchor primer, GT15A, GT15C, or GT15G. The resultant cDNA
equivalent to 50 ng of RNA was subjected to polymerase chain reaction (PCR) with 0.5
µM anchor primer, 0.5 µM arbitrary primer (10 mer kit A; Operon), 50 µM each dNTP,
1 unit of Gene Taq DNA polymerase (Nippon Gene, Toyama, Japan), and 1 unit of Taq
DNA polymerase (Takara Shuzo). PCR products were separated with 6%
polyacrylamide-8 M urea gel and analyzed by employing Vistra Fluor Imager SI
(Molecular Dynamics). The bands of interest were excised from the gel and cloned
into pSTBlue-1 Vector (Novagen). The inserted cDNA was sequenced with CEQ 2000
DNA Analysis System (Beckman Coulter) and analyzed with the BLAST program to
search the GeneBank database.
Human hepatoma cell lines (HepG2, Hep3B, HLE, HLF, HuH7, and SK-Hep1)
were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Sigma)
supplemented with 10% heat-inactivated fetal bovine serum (FBS; Atlanta Biologicals,
Norcross, Ga.) at 37 oC in a humidified atmosphere with 5% CO2 in the air12.
Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR)
Total RNAs were isolated with RNA-Bee (Biotecx Laboratories, Inc.),
followed by the treatment with RNase-free DNase (Takara Shuzo, Kyoto, Japan), and a
semi-quantitative RT-PCR analysis was performed as described previously13. The
cDNA was amplified using the sets of the primers that specifically amplify Pim family
kinases and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The sequences are
as follows; Pim-1, sense 5’-CCCGAGCTATTGAAGTCTGA-3’, antisense
5’-CTGTGCAGATGGATCTCAGA-3’; Pim-2, sense
5’-CAGTAGGGTCCCTCACCAAA-3’; Pim-3, sense
5’-CAGCGGAACCGCTCATTGCCAATGG-3’; GAPDH, sense
5’-TCCACCACCCTGTTGCTGTA-3’. The resultant PCR products were separated on
1.5% agarose gel and visualized by ethidium bromide staining. The band intensities
were measured using NIH Image Analysis Software Ver 1.61 (National Institutes of
Health, Bethesda, MD) and the ratios to GADPH were calculated.
cDNA library construction and screening
Total RNA was isolated from HepG2 cell line and polyA mRNA was separated
by OligotexTM-dT30 <Super> mRNA Purification kit (Takara Shuzo). cDNA was
synthesized with SuperScript II reverse transcriptase (Invitrogen) and oligo-dT primer,
and cDNA library was constructed in pCMVSPORT6 (Invitrogen) with Escherichia coli
DH10B (Invitrogen), according to the manufacturer’s instructions. The initial
screening was performed using GENE TRAPPER® cDNA Positive Selection System
(Invitrogen) and the oligomer, CTGTGAAGCACGTGGTGAAG, as a probe. The
obtained colonies were subjected to colony PCR screening with the sets of primers
described above. The inserted cDNA was sequenced with CEQ 2000 DNA Analysis
System (Beckman Coulter).
Northern blot analysis
Human Pim-3 mRNA expression was analyzed by using Human 12-Lane
MTNTM Blot (Clontech, Palo Alto, CA). In vitro transcribed digoxigenin-labeled
probes were hybridized overnight at appropriate temperatures (70 oC for Pim-3 and 68
oC for GAPDH). After being washed sequentially each for 15 min in 2 x and 0.5 x
SSC buffer containing 0.1% sodium dodecyl sulfate at room temperature and at 68 oC,
respectively, the hybridized probes were detected by the DIG detection kit (Boehringer
Mannheim Biochemicals), according to the manufacturer’s instructions.
Preparation of anti-Pim-3 polyclonal antibodies
Anti-Pim-3 antibodies were prepared by Asahi Techno Glass Co. (Tokyo,
Japan). Briefly, two chickens were immunized with keyhole limpet
hemocyanine-conjugated Pim-3 peptide, CGPGGVDHLPVKILQPAKAD, which
corresponds to the amino acid residues between 13 and 32 in human Pim-3 and is
conserved in murine Pim-3, and their egg yolks were harvested before and after the
immunization. IgY proteins were purified with EGGstract® IgY Purification System
(Promega) according to the manufacturer’s instructions, and they were affinity-purified
with Pim-3 peptide conjugated NHS-activated HP (Amersham Biosciences, Tokyo,
Japan). Purified antibodies were quantified by measuring the absorbance at 280 nm.
Human liver specimens were surgically obtained from the patients with their
informed consent, and mouse liver tissues were obtained from HBsTg mouse at the
indicated time intervals after splenocyte transfer. Paraffin-embedded tissue sections
were deparaffinized in xylene and rehydrated through graded concentrations of ethanol
(100% - 70%). After incubation with 0.3% hydrogen peroxide in 10 mM phosphate
buffer, pH 7.4, containing 150 mM NaCl (phosphate buffered saline; PBS (-)), sections
were incubated sequentially with 3% normal rabbit serum (DAKO, Kyoto, Japan) and
2% bovine serum albumin (BSA) in PBS (-) and with Avidin-Biotin blocking kit (Vector
Laboratories). Subsequently, the slides were treated with 10 µg/ml anti-Pim-3 IgY or
pre-immunized IgY at 4 oC overnight, followed by the incubation with 2.5 µg/ml
biotin-conjugated rabbit anti-chicken IgY antibodies (Promega) at room temperature for
30 min. The immune complexes were visualized by using the Vectastain Elite ABC kit
(Vector Laboratories) and Vectastain DAB substrate kit (Vector Laboratories) according
to the manufacturer’s instructions. The slides were counterstained with hematoxylin
(DAKO), mounted, and observed under a microscope (BX-50; Olympus, Tokyo, Japan).
Immunocytochemical analysis of HuH7 cells
Cells were cultured on Lab-Tec chamber slides (Nalge Nunc, Roskide,
Denmark). They were fixed with 4% paraformaldehyde in PBS (-) and permeated in
methanol. Then, they were blocked by incubation with 3% normal rabbit serum and
2 % BSA in PBS (-) at room temperature for 30 min, and with Avidin-Biotin blocking
kit. Subsequently, they were treated with 20 µg/ml affinity-purified anti-Pim-3 IgY or
pre-immunized IgY at 4 oC overnight, with 2.5 µg/ml biotin-labeled rabbit anti-chicken
IgY at room temperature for 30 min. The signals were amplified and visualized by the
Vectastain Elite ABC kit and Vectastain DAB substrate kit according to the
manufacturer’s instructions. The slides were counterstained with methylgreen
(DAKO), mounted, and observed under a microscope (BX-50; Olympus).
RNA interference (RNAi)
Short interfering RNA (siRNA) was synthesized with SilencerTM siRNA
Construction Kit (Ambion) according to the manufacturer’s instructions. By
employing siRNA Target Finder and Design Tool (Ambion), siRNA duplexes were
designed to target AA(N19)UU sequences in the open reading frame of mRNA encoding
Pim-3. The selected siRNA target sequence (5’-GCACGUGGUGAAGGAGCGG-3’
corresponding to 642-661) was further subjected to BLAST searches against other
human genome sequences to ensure its target specificity. We identified two distinct
cDNAs, which exhibit identity with the target sequence at 16 out of 19 nucleotides.
However, we could not detect any specific bands corresponding to these cDNAs in
HuH7 and Hep3B cell lines by RT-PCR analysis (our unpublished data), further
indicating the specificity of the used target sequence. Scramble siRNA
(5’-GCGCGCUUUGUAGGAUUCG-3’ designed by B-Bridge International, inc.) was
used as a negative control. Each siRNA duplex (final concentration 50 nM) was mixed
with 12.5 µl and 12 µl of Lipofectamine 2000 (Invitrogen) for HuH7 and Hep3B,
respectively. The mixtures were added into 2.5 ml of Opti-MEM (Invitrogen) and
allowed to stand at room temperature for 20 min. The final mixture was then added
directly into the semi-confluent cells in 6-cm culture dishes, which were washed with
serum-free DMEM beforehand. The following day, 2.5 ml of DMEM plus 20% FBS
medium was added to adjust the FBS concentration to 10%. At the indicated time
intervals, cells were harvested for further analyses.
Semi-quantitative RT-PCR analysis of siRNA transfectant
Hep3B and HuH7 cells were harvested at 2 and 4 days after the transfection,
respectively. Total RNAs were extracted and a semi-quantitative RT-PCR analysis was
performed as described above. The cDNA was amplified using the sets of the primers
that specifically amplify Pim-3 (sense
5’-ATGCTGCTCTCCAAGTTCGGCTCCCTGGCG-3’, antisense 5’-
TCCTGTGCCGGCTCGGGTCGCTCCAGCACC-3’) and GAPDH.
Cell proliferation assay
Cells were trypsinized at 2 days after the transfection, and 5 x 103 cells were
plated to each well of 96-well plate. This time point was designated as day 0. The
cell viability was determined every day using WST-1 reagent (an MTT analog from
Boehringer Mannheim Biochemicals) according to the manufacturer’s instructions.
The ratios to day 0 were calculated.
Cell cycle analysis by a flow cytometry
HuH7 cells were harvested at 4 days after the transfection and fixed with
graded concentrations of ethanol on ice. Then, they were incubated with 50 µg/ml
propidium iodide and 1 µg/ml of RNase A for 30 min at room temperature, and
quenched by adding EDTA to a final concentration of 10 µM. The filtered cells were
analyzed using a FACSCaliber (Becton Dickinson, Bedford, Mass.). The distribution
in each cell cycle phase was determined by using Cell Quest analysis software (Becton
Chromatin condensation analysis by Hoechst 33258
HuH7 and Hep3B cells were harvested at 4 days after the transfection and
stained with Hoechst 33258 in order to detect the cells with condensed nuclei under a
fluorescence microscope (BX-50; Olympus).
Identification of the genes differentially expressed in pre-malignant liver tissue
We compared the gene expression patterns between pre-malignant lesions and
normal liver tissues by employing a FDD method. The determination of the nucleotide
sequence of the resultant bands identified 24 and 19 distinct genes among the
up-regulated and down-regulated bands in pre-malignant lesions, respectively (Table 1).
Among these genes, Pim-3 expression has not been reported in normal hepatocytes.
Hence, we focused on Pim-3, a member of proto-oncogene Pim family including Pim-1
and Pim-2. A semi-quantitative RT-PCR analysis confirmed that Pim-3 mRNA
expression was significantly enhanced in the pre-malignant tissues and to a lesser
degree, in HCC tissues, compared with control (Fig. 1). In contrast, specific Pim-1
and Pim-2 transcripts were barely detected under these conditions (Fig. 1). These
results indicate that Pim-3 mRNA expression is enhanced during HCC development in
We further localized Pim-3 protein immunohistochemically in liver tissues
obtained from HBsTg mouse after splenocyte transfer. We failed to detect Pim-3
protein in unmanipulated mice (Fig. 2A) or 9 month after splenocyte transfer, when
hepatocytes with atypical nuclear configuration were not detected (data not shown).
On the contrary, Pim-3 protein was weakly detected in the cytoplasm of hepatocytes
with atypical nuclear configurations in pre-malignant lesion (Fig. 2B and E) and highly
differentiated neoplastic hepatocytes in the tumor portion7 (Fig. 2C and F). Moreover,
Pim-3 protein was detected in regenerated proliferating bile ductules (Fig. 2D, arrow),
which are assumed to be the proliferation of hepatic stem cells after the chronic liver
injury such as infection and tumor14. These results may indicate that Pim-3 protein
expression was aberrantly enhanced in liver during the course of hepatocarcinogenesis
in this model.
Cloning and determination of nucleotide sequence of human Pim-3
Because the full length human Pim-3 cDNA nucleotide sequence has not been
determined yet, we initially cloned and determined the nucleotide sequence of human
Pim-3 cDNA by screening cDNA library constructed from a human hepatoma cell line,
HepG2. Three positive clones were obtained after two rounds of screening, and these
three distinct cDNA clones contained the same insertion, which consists of 2,392 bp.
The 5’-untranslated region is 82.3 % G and C, while the 3’-untranslated region contains
5 copies of the ATTTA motif and 8 copies of TATT motif (Fig. 3A). This sequence
exhibits an identity with a partial human Pim-3 cDNA sequence predicted from EST
database (data not shown) 15. Its open reading frame encodes the protein consisting of
326 amino acids with a calculated molecular weight of 35,861 (Fig. 3A). Moreover,
the amino acid sequence of the predicted open reading frame, shares a high degree of
identity with the mouse16 and rat Pim-3 (KID-1) 8 proteins (95.0 %; Fig. 3B). Based
on these results, we judged this clone as human Pim-3 cDNA. Human Pim-3 protein
showed a high sequence identity with the quail qPim17 (73.9 %) and Xenopus Pim
(Pim-1) 18 (68.7 %) at the amino acid level (data not shown). Moreover, human Pim-3
protein shows a high sequence identity with human Pim-119 (57.1 %) and Pim-220
(44.0 %) at the amino acid level (Fig. 3C). Northern blotting analysis detected 2.4-kb
mRNA in various organs including heart, skeletal muscle, brain, spleen, kidney,
placenta, lung, and peripheral blood leukocytes (Fig. 4). In contrast, no specific band
was detected in colon, thymus, liver, and small intestine under the present experimental
conditions (Fig. 4).
Pim-3 is expressed aberrantly in human HCC
Immunohistochemical analysis failed to detect Pim-3 protein in normal liver
tissues (Fig. 5A), consistent with the Northern blotting analysis. On the contrary,
Pim-3 protein was weakly but diffusely detected in most of large regenerative nodules
and adenomatous hyperplasia, lesions with precancerous potential, which were
located adjacent to HCC areas (19 of 27 cases; Fig. 5B and 5C). Moreover, a
substantial proportion of HCC cells were immunostained with anti-Pim-3 IgY (6 of 27
cases; Fig. 5D and 5E) but not the pre-immunized IgY (data not shown). Furthermore,
Pim-3 protein was observed markedly in regenerated proliferating bile ductules (27 of
27 cases; Fig. 5C and 5F, arrows). Because the staining patterns were similar to that
observed in HBsTg mouse model (see Fig 2), these results would indicate that Pim-3
protein expression was aberrantly enhanced in precancerous lesion, also in humans, and
a portion of HCC cells.
Constitutive Pim-3 expression in human hepatoma cell lines
Immunohistochemical analysis indicated that Pim-3 protein expression was
aberrantly enhanced not only in precancerous lesion but also in a portion of HCC cells
in human HCC tissues (Fig. 5). This finding prompted us to examine Pim-3
expression in human hepatoma cell lines, by RT-PCR. To exclude the possibility that
contaminated genomic DNA gave rise to the generation of the amplified bands, we used
total RNA samples that were treated with DNase. Under the present condition, Pim-3
transcript was detected in all hepatoma cell lines, whereas no specific band was detected
in the normal liver tissue (Fig. 6A), consistent with the Northern blotting analysis. The
exclusion of reverse transcriptase failed to give rise to any bands, further indicating the
specificities of RT-PCR (Fig. 6A non-RT). Moreover, an immunocytochemical
analysis detected immunoreactive Pim-3 proteins in HuH7 cell line, when incubated
with anti-Pim-3 antibodies (Fig. 6B-a) but neither pre-immunized IgY (Fig. 6B-b) nor
anti-Pim-3 adsorbed with the relevant peptide (Fig. 6B-c). Immunoreactive Pim-3
proteins were similarly detected in all six human hepatoma cell lines, consistent with
RT-PCR analysis (data not shown). Collectively, these results would indicate that
Pim-3 was constitutively expressed in human hepatoma cell lines.
RNAi ablation of Pim-3 induces cell death to hepatoma cell lines
Because Pim-1 and Pim-2 were required to induce cell cycle progression21, 22
and anti-apoptotic effects21-25, we next examined the role of Pim-3 in cell proliferation,
by ablating endogenous Pim-3 mRNA expression in HuH7 and Hep3B cell lines with
RNAi. Endogenous Pim-3 mRNA level was decreased after the transfection with
specific Pim-3 siRNA but not Scramble siRNA (Fig. 7A). Under these conditions,
transfection with Pim-3 siRNA significantly retarded cell proliferation, compared with
Scramble siRNA-transfected and the control cells (Fig. 7B and C). These results
suggested that Pim-3 ablation has adverse effects on the proliferation of hepatoma cell
lines. We further observed that HuH7 cells detached from the plates later than 4 days
after the transfection with Pim-3 but not Scramble siRNA (Fig. 8A). Moreover, Pim-3
siRNA transfectants exhibited a higher ratio of sub-G1 populations with reduced G1 and
G2/M populations, compared with Scramble siRNA transfectants and control cells (Fig.
8B). Furthermore, the proportion of cells with condensed nuclei was significantly
higher in both HuH7 and Hep3B cells transfected with Pim-3 siRNA, than those
transfected with Scramble siRNA (Fig. 8C). These observations would indicate that
the ablation of Pim-3 might induce apoptosis in these hepatoma cell lines.
Transcriptome analysis has been widely applied to elucidate molecular
mechanisms of various types of diseases and can provide many important clues,
particularly for understanding the molecular pathogenesis of oncogenesis, where the
expression of many genes changes simultaneously26, 27. Several independent groups
performed transcriptomal studies on human HCC28-33. However, in most studies, the
gene expression pattern was compared between tumor and non-tumor portions obtained
from the same patients28-33. Because these non-tumor portions exhibit usually
hepatocyte dysplasia, this type of analysis may fail to detect the changes in gene
expression that have already existed at the stage of hepatocyte dysplasia. In order to
circumvent these pitfalls, we compared gene expression patterns between pre-malignant
lesions and normal tissues by using FDD. We observed that various genes were
selectively changed in pre-malignant lesions. Moreover, a semi-quantitative RT-PCR
analysis did not detect any significant differences in the expression of several of these
genes between malignant and pre-malignant lesions (our unpublished data), supporting
our assumption that the changes in gene expression which have already existed at the
stage of hepatocyte dysplasia, might be undetected in the preceding studies.
Among the genes identified in this study, we focused on Pim-3. Pim-3 was
originally identified as depolarization-induced gene KID-1 in PC12 cell line, a rat
pheochromocytoma cell line8. Subsequently, several independent groups observed a
selective expression of its mRNA in neuronal system16, 34, 35, but not liver. By using
human Pim-3 cDNA as a probe, we detected Pim-3 mRNA in several organs such as
brain and spleen, but not liver. On the contrary, Pim-3 mRNA expression was detected
in all human hepatoma cell lines that we examined. Moreover, immunohistochemical
analysis detected immunoreactive Pim-3 protein in precancerous lesions and a portion
of HCC cells. Furthermore, Pim-3 protein was also detected in regenerating bile
ductules, which are assumed to be the proliferation of hepatic stem cells after the
chronic liver injury such as infection and tumor14. Liver cell destruction and
regeneration are thought to prepare the mitogenic, mutagenic environment, and
impaired liver regeneration leading to HCC development36. Thus, Pim-3 expression
may aberrantly be enhanced from hepatocyte regeneration process to its malignant
Deneen and colleagues provided evidence on the crucial involvement of Pim-3
in EWS/ETS-mediated malignant transformation of mouse NIH 3T3 cells37. They
demonstrated that Pim-3 was a common transcriptional target of EWS/ETS.
EWS/ETS fusion proteins retain an intact ETS DNA-binding domain and can bind to a
binding sequence in the target genes through this domain38. Thus, Pim-3 gene
transcription may be regulated not only by EWS/ETS fusion proteins but also other Ets
family proteins. Several independent groups reported that Ets-1, one of the Ets family
proteins, was expressed in human HCC tissues39, 40. Ito and colleagues described that
Ets-1 expression was markedly enhanced in non-cancerous lesions adjacent to HCC
lesions and suggested that Ets-1 had a crucial role in hepatocarcinogenesis and HCC
progression during their early phases39. In line with these observations, we also
observed that another transcription factor with an ETS-domain, polyomavirus enhancer
A binding protein-3, was expressed selectively in HCC and that polyomavirus enhancer
A binding protein-3 induced constitutive gene expression of a pro-angiogenic factor,
interleukin (IL)-8, in HCC41. Thus, it is tempting to speculate that a transcription
factor(s) with an ETS-domain, may induce ectopic Pim-3 gene expression in liver,
during the course of hepatocarcinogenesis.
Several lines of evidence demonstrated that the gene expression of Pim-1 and
Pim-2 could be regulated by IL-6-gp130-mediated signal transducers and activators of
transcription (STAT) family protein, STAT342, 43. STAT3 signals can advance cell
cycles and prevent apoptosis by inducing Pim-1 and c-Myc in lymphomagenesis43. In
the liver, IL-6-deficient mice exhibited an impaired liver regeneration after a partial
hepatectomy44. Several lines of evidence have revealed that Bcl-xL expression is
up-regulated by IL-6-gp130-mediated STAT3 and prevents hepatocyte apoptosis45, 46,
and that the constitutive activation of STATs observed during oncogenesis can cause a
permanent alteration in the genetic program46, 47. These observations suggest that
STAT3 signals could regulate hepatocyte regeneration also during the course of
HBV-induced hepatocarcinogenesis. We observed that Pim-3 gene ablation by RNAi
attenuated proliferation rates and caused cell death in hepatoma cell lines. Thus, if
Pim-3 was also regulated by STAT3, these observations suggest that Pim-3 would also
be involved in STAT3-mediated prevention of apoptosis and/or cell cycle progression.
Pim-1 and Pim-2 are also known as proto-oncogene to be involved in
lymphomagenesis48, 49. Because Pim-1 and Pim-2 can induce anti-apoptotic effects21-25,
Pim-3 may be involved in cell cycle regulation and/or anti-apoptosis. We also
observed that gene ablation of Pim-3 caused cell death to human hepatoma cell lines,
later than 3 days after the transfection. These results suggest that Pim-3 can regulate
cell cycle and/or apoptosis process indirectly by phosphorylating a molecule(s)
upstream in these processes. Although Pim-1 can phosphorylate several molecules
such as Cdc25A50, a G1/S cell cycle regulator, Pim-3 could not interact with Cdc25A37.
Pim-1 can also phosphorylate valosine containing protein (VCP)/p9742, 43, a mammalian
homolog of Saccaromyces cerevisiae Cdc48p. Pim-1 can up-regulate further the
expression of an anti-apoptotic molecule, Bcl-2 and Bcl-xL, by augmenting the
expression of VCP42, 43. In HCC tissues and human hepatoma cell lines, evidence is
accumulating to indicate that Bcl-xL is constitutively expressed and is a major executer
to prevent apoptosis51-53. Moreover, VCP was also detected in human HCC tissues54.
If VCP could be phosphorylated by Pim-3 as well as Pim-1, Pim-3 may exert an
anti-apoptosis effect by augmenting indirectly the expression of an anti-apoptotic
molecule, similarly as Pim-1. Moreover, if the contents of target molecules may differ
between HuH7 and Hep3B cell lines, these may account for different patterns of the
effects of Pim-3 gene ablation on the proliferation of these cell lines.
The kinase activity of Pim-3 was crucially involved in EWS/ETS-mediated
malignant transformation of mouse NIH 3T3 cells37. Our present observations suggest
that Pim-3 can regulate anti-apoptosis process and/or cell cycle progression probably by
modulating molecules involved in these processes. Accumulating evidence indicated
that Pim-3 can auto-phosphorylate itself8, 17, but it still remains elusive on physiological
substrates of Pim-3. The identification of a substrate(s) may shed novel light on
Pim-3-mediated regulatory mechanisms of apoptosis and/or cell cycle progression.
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Identification of genes differentially expressed in pre-malignant lesion of HBsTg mice
classification description accession number
transcription factors Y box protein 3 AK029441
immune system proteins complement component 3 BC043338
xenobiotic metabolism ceruloplasmin NM_00775
metallothionein II AK002567
oncogenes 24p3 (lipocalin 2) X81627
metabolism enzymes aldehyde dehydrogenase family 1, subfamily A1 BC044729
hemoglobin α, adult chain 1
phosphoenolpyruvate carboxy-kinase 1 NM_011044
succinate dehydrogenase complex, subunit A flavoprotein BC031849
growth factors, cytokines, and chemokines growth differentiation factor 15 (macrophage inhibiting compound-1) NM_011819
insulin-like growth factor binding protein 1 NM_008341
non-receptor protein kinases serine/threonine kinase pim-3 NM_145478
not classified betaKlotho AF178429
pol protein XM_196572
serine (or cysteine) proteinase inhibitor, clade A, member 6 NM_007618
putative proteins hypothetical Esterase/acetylhydrolase structure containing protein NM_026347
putative e1 protein AK090127
similar to bile acid Coenzyme A: amino acid N-acyltransferase NM_145368
unknown fibronectin non-coding region
hypothetical protein non-coding region chromosome 5
immune system proteins
complement component C1SA AF459017
extracellular transport/carrier proteins serum amyloid A-1 M13521
metabolism enzymes cytochrome P450 4A10 BC031141
hemoglobin β, adult major chain
stearoyl-Coenzyme A desaturase 1 BC007474
isocitrate dehydrogenase 1 (NADP+), soluble AK087063
not classified endogenous retrovirus 3' LTR K02892
glutatione S-transferase BC009805
group1 major urinary protein X03208
major urinary protein 1 BC012221
major urinary protein 2 BC012259
major urinary protein 3 XM_135398
major urinary protein 11 and 8 AK011413
preimplantation protein 2 AK028563
ubiquitin-associated protein 1 NM_023305
mitchondrial gene cytochrome oxidase A1 V00711
putative proteins archerase AY071852
dis3 protein homolog AK032091
FIGURE 1. Semi-quantitative RT-PCR analysis for proto-oncogene Pim family
mRNA expression in HBsTg mice.
A. Total RNAs were extracted from HBsTg mice before (symbol N), 9 (whole liver,
symbol 9), 15 months (pre-malignant lesions, symbol 15) after splenocyte transfer, or
transgenic splenocytes transfer (symbol C).
B. Total RNAs were extracted from pre-malignant (symbol P) or malignant (symbol
M) tissues of HBV transgenic mice 15 months after splenocyte transfer.
Representative results from three independent experiments are shown in the upper
panels. The ratios of the PCR product for Pim-3 to GAPDH were determined, and
relative intensities were calculated to assume the ratio of untreated mice as 1.0. Then,
means and SD were calculated and are shown in the lower panels. Statistical
significance was evaluated using ANOVA test, and p < 0.05 was accepted as statistically
significant. *, p < 0.05 compared with N.
FIGURE 2. Immunohistochemical analysis of Pim-3 in HBsTg mice. HBsTg mice
liver tissues before (A) and 15 months after splenocyte transfer (B, D, and E, non-tumor
portions; C and F, tumor potions) were immunostained by anti-Pim-3 IgY as described
in Materials and Methods. Representative results are shown here.
B. Pre-malignant lesion is indicated with arrowheads.
C. Tumor portion is indicated with arrowheads.
D. The positively stained regenerated proliferating bile ductule. The arrow indicates
regenerated proliferating bile ductule.
E and F. The positively stained hepatocytes at a higher magnification of the square in
B and C.
Original magnification; A to C, x 100; D to F, x 400. Scale bars are 50 µm.
FIGURE 3. A. Structure of human Pim-3 cDNA. The nucleotide and predicted
amino acid sequences of human Pim-3 are shown. The nucleotide sequence is
numbered. The predicted amino acid sequence is shown in a single-letter code below
the nucleotide sequence. The AT-rich motifs are indicated in boxes and highlights.
The region used as the probe for Northern blot analysis is underlined.
B. and C. Amino acid alignment of Pim family proteins. The amino acid sequences
of human, rat, and mouse Pim-3s (B) or other members of human Pim family kinases
(C) were aligned using DNASIS-Mac version 3.0 software (Hitachi Software
Engineering Co., Ltd., Yokohama, Japan). The residues identical to human Pim-3 are
FIGURE 4. Human Pim-3 mRNA expression in human normal tissues. Northern
blot analysis was performed as described in Materials and Methods, and representative
results are shown here. sk. muscle, skeletal muscle; PBL, peripheral blood leukocytes.
GAPDH mRNA expression was analyzed in parallel to evaluate the amount of mRNA
loaded in each lane.
FIGURE 5. Immunohistochemical analysis of Pim-3 in HCC tissues. Human normal
liver tissue (A) or HCC tissues (B to F) were immunostained by anti-Pim-3 IgY as
described in Materials and Methods. Representative results are shown here.
B. Precancerous lesions. The lesions surrounded with arrowheads and arrows are
precancerous lesions and HCC lesions, respectively.
C. Precancerous lesion in the square of B is shown at a higher magnification.
Arrows indicate regenerated proliferating bile ductules.
D and E. The positively stained HCC cells. E indicates the square in D at a higher
F. The positively stained regenerated proliferating bile ductules at a higher
magnification of the square in D. Arrows indicate regenerated proliferating bile
Original magnification; A, B and D, x 100; C, E, and F, x 400. Scale bars are 50 µm.
FIGURE 6. A. Pim-3 mRNA expression in various human hepatoma cell lines.
Total RNAs were extracted from human hepatoma cell lines and normal liver tissue.
RT-PCR was performed, and representative results from three independent experiments
are shown here. Analysis for Pim-3 expression was performed without reverse
transcriptase treatment and the results are shown as non-RT.
B. Pim-3 protein expression in HuH7 cells. HuH7 cells were immunostained by
anti-Pim-3 IgY (B-a), by pre-immunized IgY (B-b), or by anti-Pim-3 antibodies
absorbed with the relevant peptide (B-c) as described in Materials and Methods.
Representative results from three independent experiments are shown here. Original
magnification, x 400. Scale bars are 50 µm.
FIGURE 7. The effects of endogenous Pim-3 ablation on cell proliferation.
A. Semi-quantitative RT-PCR analysis for Pim-3 mRNA levels in siRNA transfected
cells. Total RNAs were extracted from the transfectant with Pim-3 siRNA (symbol P),
Scramble siRNA (symbol S), or no siRNA (symbol N) 2 (Hep3B) or 4 days (HuH7)
after the transfection as described in Materials and Methods.
B and C. Cell proliferation rates were determined on HuH7 (B) and Hep3B (C) cells
transfected with Pim-3 siRNA (circles), Scramble siRNA (squares), or no siRNA
(triangles) by WST-1 assay. Cells were trypsinized and 5 x 103 cells were plated to
each well of 96-well plate at 2 days after the transfection. This time point was
designated as day 0. The ratios to day 0 were calculated. Results are expressed as
means (n = 3), and error bars indicate SD. Representative results from three
independent experiments are shown here. Statistical significance was evaluated using
ANOVA test, and p< 0.05 was accepted as statistically significant. *, p < 0.05
compared with no siRNA samples at the same time point.
FIGURE 8. The effects of endogenous Pim-3 ablation on apoptosis in HuH7 cells.
A. HuH7 cells were observed under an inverted microscope at 4 days after the
transfection with Pim-3 or Scramble siRNA under the same conditions as FIG. 7.
Representative results from three independent experiments are shown here. Original
magnification, x 200. Scale bars are 50 µm.
B. Cell cycles were analyzed at 4 days after the transfection, by using a flow
cytometry as described in Materials and Methods. Representative results from three
independent experiments are shown here.
C. Chromatin condensation was analyzed at 4 days after the transfection as described
in Materials and Methods. Representative results from three independent experiments
are shown here. Results are expressed as means (n = 6), and bars indicate SE.
Statistical significance was evaluated using ANOVA test, and p < 0.05 was accepted as
statistically significant. *, p < 0.05 compared with Scramble siRNA samples.
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