Isolation and Characterization of a Novel Oncogene,
Amplified in Liver Cancer 1, within a Commonly
Amplified Region at 1q21 in Hepatocellular Carcinoma
Ning-Fang Ma,1,2* Liang Hu,2* Jackie M. Fung,2 Dan Xie,4 Bo-Jian Zheng,3 Leilei Chen,2 Dong-Jiang Tang,2 Li Fu,2
Zhenguo Wu,5 Muhan Chen,2 Yan Fang,4 and Xin-Yuan Guan2,4
Amplification of 1q21 is the most frequent genetic alteration in human hepatocellular carcinoma
(HCC), being detected in 58%-78% of primary HCC cases by comparative genomic hybridization. Recently, we isolated a candidate oncogene, Amplified in Liver Cancer 1 (ALC1), from 1q21
by hybrid selection. Here we demonstrate that ALC1 was frequently amplified and overexpressed
in HCC. ALC1-transfected cells possessed a strong oncogenic ability, increasing the colony
formation in soft agar and increasing the tumorigenicity in nude mice, which could be effectively
suppressed by small interfering RNA against ALC1. Functional studies showed that overexpression of ALC1 could promote G1/S phase transition and inhibit apoptosis. Molecular studies
revealed that the oncogenic function of ALC1 might be associated with its roles in promoting cell
proliferation by down-regulating p53 expression. Conclusion: These results suggest that ALC1 is
the target oncogene within the 1q21 amplicon and plays a pivotal role in HCC pathogenesis.
(HEPATOLOGY 2008;47:503-510.)
H
epatocellular carcinoma (HCC) is one of the
most frequently diagnosed human cancers
worldwide with a very poor prognosis. It is believed that HCC, like many other solid tumors, develops
Abbreviations: ALC1, amplified in liver cancer 1; BAC, bacterial artificial
chromosome; Bax, BCL2-associated X protein; CDK, cyclin-dependent kinase;
cDNA, complementary DNA; CHD1, chromodomain helicase DNA binding protein 1; FISH, fluorescence in situ hybridization; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HCC, hepatocellular carcinoma; HELICc, helicase
superfamily c-terminal domain; IHC, immunohistochemistry; PCR, polymerase
chain reaction; siRNA, small interfering RNA; STS, straurosporine; TMA, tissue
microarray; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nickend labeling.
From the 1Department of Histology and Embryology, Guangzhou Medical College, Guangzhou, China; Departments of 2Clinical Oncology and 3Microbiology,
University of Hong Kong, Pokfulam, Hong Kong; 4State Key Laboratory of Oncology in Southern China, Cancer Center, Sun Yat-Sen University, Guangzhou, China; and 5Department of Biochemistry, Hong Kong University of Science and
Technology, Hong Kong.
Received August 7, 2007; accepted September 28, 2007.
Supported by a Research Grant Council grant (HKU 7393/04M), the Research
Fund for the Control of Infectious Diseases (02040162), the Hundred Talents
Program of Sun Yat-Sen University (85000-3171311), the Leung Kwok Tze
Foundation, and the Foundation of the Guangzhou Science and Technology Bureau
(2005Z1-E0131).
*These authors contributed equally to this study.
Address reprint requests to: Xin-Yuan Guan, Ph.D., Department of Clinical
Oncology, University of Hong Kong, Room L10-56, Laboratory Block, 21 Sassoon
Road, Hong Kong. E-mail: [email protected].
Copyright © 2007 by the American Association for the Study of Liver Diseases.
Published online in Wiley InterScience (www.interscience.wiley.com).
DOI 10.1002/hep.22072
Potential conflict of interest: Nothing to report.
from the accumulation of alterations of cancer-related
genes critical to processes such as cell proliferation, apoptosis, and other functions. It has been estimated that
approximately three to six genetic events are necessary to
transform a normal cell into a cancer cell.1 Amplification
of 1q is one of the most frequent genetic alterations in
primary HCC, being detected in 58%-78% of HCC patients by comparative genomic hybridization.2-5 A minimal amplified region has since then been narrowed down
to 1q21,4,6 and this suggests the existence of an oncogene
at 1q21 that plays an important role in HCC pathogenesis. Amplification of 1q has also been frequently detected
in many other solid tumors, including bladder,7 breast,8
nasopharyngeal carcinoma,9 and esophageal tumors.10
Therefore, the identification of the target gene responsible
for the 1q21 amplification event is imperative for understanding the molecular mechanism of cancer development in many solid tumors including HCC.
Hybrid selection of chromosome region–specific
transcripts using microdissected DNA is a rapid and
effective method to isolate amplified genes from an
amplicon.11,12 Recently, we isolated a novel candidate
oncogene from 1q21 named Amplified in Liver Cancer
1 (ALC1; Gene Bank accession no. AF537213), using
this strategy. Sequencing analysis showed that this gene
belongs to the sucrose nonfermenting 2 (SNF2)-like
family, containing putative helicase motifs. In the
present study, we demonstrated that ALC1 was ampli503
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fied and overexpressed in over 50% of primary HCC
patients. The oncogenic function of ALC1 was demonstrated by both in vitro and in vivo assays. The molecular mechanism of ALC1 in tumorigenesis has been
associated with its role in promoting G1/S transition
and inhibition of apoptosis.
Materials and Methods
HCC Samples and HCC Cell Lines. Primary HCC
specimens were obtained with informed consent from patients who underwent hepatectomy for HCC at the Cancer
Center of Sun Yat-Sen University (Guangzhou, China). Human liver cell line LO2 and HCC cell lines BEL7402, QSG7701, QGY-7703, PLC8024, CRL8064, and HepG2 were
obtained from the Institute of Virology of the Chinese Academy of Medical Sciences (Beijing, China). H2M and H4M
were previously established in our laboratory.13 QSG-7701,
QGY-7703, PLC8024, and CRL8064 are hepatitis B virus–
positive cell lines, whereas LO2, BEL7402, HepG2, H2M,
and H4M are hepatitis B virus–negative cell lines.
Chromosome Microdissection, Hybrid Selection,
and Fluorescence In Situ Hybridization (FISH).
Chromosome microdissection and polymerase chain reaction (PCR) amplification of microdissected DNA were
performed as described previously.11 Briefly, five copies of
the 1q21 band were dissected and amplified by PCR with
UN1 primer. Hybrid selection was performed as described previously.12 Briefly, 5 ␮g of PCR products of
microdissected DNA was immobilized on a nylon membrane and hybridized with complementary DNA
(cDNA) prepared from an HCC case (H-4) containing
1q21 amplification. After a stringent wash, specifically
hybridized cDNA was eluted, recovered by PCR, and
analyzed by sequencing analysis. Bacterial artificial chromosome (BAC) clones along 1q, including the BAC containing the ALC1 gene (RP11-337C18), were selected for
interphase FISH study. BAC DNA was labeled and then
hybridized to interphase nuclei by FISH according to the
method described previously.12
Construction of Tissue Microarray (TMA) and Immunohistochemistry (IHC). A total of 320 formalinfixed and paraffin-embedded HCC tissue specimens
were selected from the Cancer Center of Sun Yat-Sen
University. An HCC TMA was constructed as described previously.14 Five-micrometer consecutive sections of a microarray block were made with a
microtome. IHC studies were performed with the standard streptavidin-biotin-peroxidase complex method.
TMA sections were deparaffinized and incubated with
polyclonal anti-ALC1 antibody (Boster Biotechnology
HEPATOLOGY, February 2008
Co., Ltd., Wuhan, China) in a dilution of 1:100 at 4°C
overnight.
Tumorigenic Ability of ALC1. To evaluate the tumorigenic ability of ALC1, ALC1 was cloned into expression vector pcDNA3.1(⫹) (Invitrogen, Carlsbad, CA)
and transfected into mouse fibroblast cell line NIH3T3,
immortalized liver cell line LO2, and HCC cell line
QGY-7703. A soft agar colony formation assay was carried out by the suspension of 1 ⫻ 104 cells in 0.4% Seaplaque agar and seeded onto solidified 0.6% agar in a
6-well plate. Colonies that were at least 4 times as large as
the original single cell were counted at day 21. Triplicate
independent experiments were performed. Tumor formation in nude mice was performed with a single injection of
4 ⫻ 106 cells. Empty vector–transfected cells were injected into the left dorsal flank, and ALC1-transfected
cells were injected into the right dorsal flank of the same
animal. The animals were examined for tumor formation
over a period of 1 month.
Detection of DNA Content by Flow Cytometry.
ALC1-transfected and vector-transfected QGY-7703 cells
were cultured in Dulbecco’s modified Eagle’s medium
containing 10% fetal bovine serum. Serum was withdrawn from the culture medium when cells were 70%
confluent. After 72 hours, 10% fetal bovine serum was
added to the medium for an additional 12 hours. Cells
were fixed in 70% ethanol, stained with propidium iodide, and analyzed by a flow cytometer. Triplicate independent experiments were performed.
Small Interfering RNA (siRNA) Transfection.
H2-M cells were transfected with double-stranded siRNAs
(Ambion, Inc., Austin, TX) with Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions. Forty-eight hours after transfection, the gene-silencing
effect was measured by reverse-transcription PCR. Three independent experiments were performed.
Western Blotting Analysis. Western blot analyses
were performed with the standard method with antibodies to ALC1 (Boster Biotechnology), p53, cyclin E,
caspase 3, BCL2-associated X protein (Bax), ␤-actin
(Santa Cruz Biotechnology, Santa Cruz, CA), p21, and
cyclin-dependent kinase (cdk2; Cell Signaling Technology, Beverley, MA). The densitometry data were analyzed
with Scion Image software (version beta 3b, Scion Corp.,
Frederick, MD).
Detection of Apoptosis by Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick-End Labeling
(TUNEL) Assay. ALC1-transfected and vector-transfected QGY-7703 cells were treated with straurosporine
(STS; 1 ␮M) for 4 hours. Morphological changes in the
nuclear chromatin undergoing apoptosis were detected by
TUNEL assay according to the manufacturer’s protocol
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505
(Roche, Mannheim, Germany). Triplicate independent
experiments were performed.
Statistical Analysis. The comparison of ALC1-transfected and vector-transfected cells in anchorage-independent growth was ascertained by the Student t test. The
significance between the tumor size induced by ALC1transfected QGY-7703 cells and vector-transfected cells
in tumor xenograft experiments was determined by the
Student t test. The difference of the percentage of S phase
cells between ALC1-transfected and vector-transfected
QGY-7703 cells in serum-stimulation experiments was
analyzed by the Student t test. In TUNEL assay, the difference of the apoptotic index between ALC1-transfected
and vector-transfected QGY-7703 cells was compared by
the Student t test. P values of ⬍0.05 were considered to be
significant.
Results
Isolation of ALC1. To identify the most frequently
amplified region at 1q, amplification frequencies in 10
different regions along 1q were studied by interphase
FISH with BAC clone probes in 60 primary HCC specimens. The results showed that 1q21 was the most frequently amplified region, being detected in 36/60 (60%)
HCC specimens (Fig. 1A). To isolate the target oncogene
within the 1q21 amplicon, microdissected DNA from
1q21 was used to select region-specific transcripts from a
cDNA library generated from a primary HCC case with
1q21 amplification. An amplified DNA probe was specifically hybridized to 1q21 by FISH (Fig. 1B) and then used
to select 1q21-specific transcripts from a cDNA library
generated from a primary HCC case (H-4) with 1q21
amplification. With this strategy, a candidate oncogene,
ALC1, was isolated, and a BAC clone (RP11-337C18)
containing ALC1 was mapped to 1q21 (Fig. 1C). FISH
with the BAC probe to H-4 cells demonstrated that ALC1
was amplified in H-4 (Fig. 1D).
The open reading frame of the ALC1 gene was cloned
into a green fluorescent protein– expressing vector, and
expression of the ALC1 protein was localized to the nucleus (Fig. 1E). The full-length messenger RNA of ALC1
consists of 2980 base pairs with a putative open reading
frame coding an 897aa protein. Sequencing analysis
showed that ALC1 belongs to the SNF2-like family, containing a conserved SNF2_N domain, a helicase superfamily domain [helicase superfamily c-terminal domain
(HELICc)], and a Macro domain (Fig. 1F). The
SNF2_N domain is composed of 280 amino acids, and
the sequence homology between the SNF2_N domains of
ALC1 and another SNF2-like family member, chromodomain helicase DNA binding protein 1 (CHD1), is
Fig. 1. Isolation of ALC1 from 1q21 amplicon. (A) Amplification of
different regions along chromosome 1q in 60 HCC cases was detected
by FISH. The number of cases amplified is indicated above each bar. (B)
FISH analysis showed that the amplified microdissected DNA probe was
specifically hybridized to normal chromosome 1q21 (red signals). This
microdissected DNA was used for cDNA selection from an HCC case with
1q21 amplification. (C) ALC1 was mapped to 1q21 by FISH with a BAC
clone containing ALC1 (indicated by arrows). (D) A representative example of ALC1 gene amplification detected in H-4 cells by FISH with the BAC
probe (red signals). A BAC probe from 1p32 (green signals) was used as
a control. (E) ALC1 was cloned to pEGFP vector. The exogenously
expressed ALC1-EGFP fusion protein (green color) was sublocalized in
the nucleus. (F) Compared to CHD1, the predicted protein structure of
ALC1 also has SNF2_N and HELICc domains.
45% identical. The sequence homology of the HELICc
domain (containing 107aa) between ALC1 and CHD1 is
59% identical.
Amplification and Overexpression of ALC1 in
HCC. Amplification of ALC1 was studied by FISH with
the BAC containing the ALC1 gene with a TMA containing 320 primary HCC specimens. Amplification of ALC1
was detected in 86/170 (50.6%) informative cases (Fig.
2A). Overexpression of ALC1 in protein level in the same
TMA was investigated by IHC with anti-ALC1 antibody.
The specificity of the antibody was tested by western blotting, and a 98-kD protein was detected (Fig. 2B). In comparison with their matched nontumor liver counterparts,
ALC1 overexpression was found in 163/311 (52.4%) in-
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Fig. 2. Overexpression of ALC1 in
primary HCCs and HCC cell lines. (A)
A representative example of ALC1
amplification in HCC TMA detected
by FISH with the BAC clone containing ALC1. (B) A 98-kD protein was
detected by anti-ALC1 antibody. (C)
Positive nuclear staining of ALC1
was frequently detected in primary
HCC (left) but not in its matched
adjacent nontumor tissue specimen
(right) by IHC. (D) The IHC result in
TMA was verified on a larger tissue
section containing HCC tissue (upper
part) and surrounding nontumor liver
tissue (lower part). RNA expression of
ALC1 was tested in (E) primary HCC
cases and(F) HCC cell lines by northern blot analysis. In primary HCC,
ALC1 expression was compared between tumors (T) and their matched
nontumor liver tissues (N).
formative HCC cases (Fig. 2C). The IHC results in TMA
were verified with larger tissue sections containing HCC
tissues and their surrounding nontumor liver tissues (Fig.
2D). Overexpression of ALC1 in RNA level was studied
by northern blot analysis in 24 primary HCCs and 8
HCC cell lines. The results showed that the overexpression of ALC1 was observed in 13/24 (54.2%) primary
HCCs (Fig. 2E) and 7/8 HCC cell lines (Fig. 2F).
Tumorigenic Ability of ALC1. To determine the tumorigenic potential of ALC1, the full-length cDNA of the
gene was cloned into expressing vector pCDNA3.1 and
stably transfected into human liver cell line LO2 and
HCC cell line QGY-7703 cells. The expression level of
ALC1 in transfected cells was determined by northern
blot hybridization (Fig. 3A). The tumorigenic ability of
ALC1 was studied by anchorage-independent growth in
soft agar and tumor formation in nude mice. As shown in
Fig. 3B, ALC1-transfected LO2 and QGY-7703 cells
were able to form more colonies in soft agar in comparison with blank vector–transfected cells (P ⬍ 0.05). Because QGY-7703 is an HCC cell line, it shows a stronger
colony formation ability than immortalized liver cell line
LO2.
Tumor xenograft experiments in nude mice demonstrated that ALC1 could dramatically increased tumorigenicity of LO2 and QGY-7703 cells in tested animals.
Tumor formation was observed in 4/6 and 0/6 ALC1transfected and blank vector–transfected LO2 cells, re-
spectively (Fig. 3C). For QGY7703 cells, tumor
formation was found in 12/12 and 4/12 ALC1-transfected and blank vector–transfected cells, respectively. In
addition, the tumor size in ALC1-transfected QGY-7703
cells (average tumor volume: 464 mm3) was significantly
larger than that of vector-transfected cells (average tumor
volume: 88 mm3, Student t test, P ⬍ 0.001).
Overexpression of ALC1 Promotes G1/S Phase
Transition. To characterize the molecular mechanism of
ALC1 in HCC development, the role of ALC1 in the cell
cycle was investigated. Following synchronization of cells
at the G1 phase by serum starvation for a period of 3 days,
G1/S phase transition in ALC1-transfected and blank vector–transfected QGY-7703 cells was stimulated by the
addition of serum to the culture medium. DNA content
was analyzed by flow cytometry, and the results indicated
that ALC1 could facilitate DNA synthesis and promote
G1/S phase transition (Fig. 3D). The percentages of cells
in G1 and S phases were similar between ALC1-transfected and vector-transfected QGY-7703 cells when they
were cultured in 10% serum and during the serum starvation. However, the percentages of cells in the S phase
were significantly higher in ALC1-transfected QGY-7703
cells (37.5 ⫾ 2.2%) than in vector-transfected
QGY-7703 cells (25.7 ⫾ 1.8%) 12 hours after serum
stimulation (P ⬍ 0.05).
Inhibition of ALC1 Expression by RNA Interference. HCC cell line H2-M, which expresses a high level
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significantly reduced in ALC1-si1–treated cells (22.9 ⫾
1.9%) compared with that in control-si–treated cells
(34.7 ⫾ 2.1%, P ⬍ 0.05).
ALC-1 Down-Regulates p53 Expression. To reveal
the mechanism of ALC-1 in promoting G1/S transition,
the expression of p53 between ALC1-transfected and vector-transfected QGY-7703 cells was compared. The result
indicated that p53 expression was down-regulated (Fig.
4D). Furthermore, other members of the p53 pathway,
including p21cip1, cdk2, and cyclin E, were studied. The
results showed that p21Waf1/cip1 was down-regulated
whereas Cdk2 and cyclin E were up-regulated (Fig. 4D)
in ALC1-transfected QGY-7703 cells.
Overexpression of ALC1 Inhibits Apoptosis. The
potential role of ALC1 in apoptosis was tested by the
treatment of ALC1-transfected and vector-transfected
QGY-7703 cells with STS, a broad-spectrum kinase
inhibitor that can induce apoptosis in a wide variety of
cells. Prior to STS treatment, the apoptotic index was
found to be similar between ALC1-transfected and vector-transfected QGY-7703 cells, but following the
treatment of cells with STS for 4 hours, the apoptotic
index was significantly higher in vector-transfected
QGY-7703 cells (65%) than that of ALC1-transfected
QGY-7703 cells (23%, P ⬍ 0.01) (Fig. 5A,B). Apoptosis-associated proteins, caspase 3 and Bax, were
tested and compared between ALC1-transfected and
vector-transfected QGY-7703 cells prior to and following STS treatment, and the results showed that expression of both caspase 3 and Bax was down-regulated in
ALC1-transfected QGY-7703 (Fig. 5C).
Fig. 3. Oncogenic ability of ALC1. (A) Expression of ALC1 in ALC1transfected LO2 and QGY-7703 cells detected by northern blot hybridization. Blank vector–transfected cells were used as controls. (B) Rates of
colony formation in soft agar detected in ALC1-transfected and blank
vector–transfected LO2 and QGY-7703 cells (**P ⬍ 0.05). (C,D) Representative examples of tumors formed in nude mice following injection
of ALC1-expressing LO2 cells (left) and QGY-7703 cells (right). ALC1expressing cells and mock cells were injected into the right and left dorsal
flanks, respectively. (E) Flow cytometry histogram showing that overexpression of ALC1 in ALC1-expressing QGY-7703 cells could promote
G1/S phase transition compared to vector-transfected QGY-7703 cells.
of ALC1, was used in the siRNA experiment. Three siRNAs targeting ALC1 were tested for their efficiency of
ALC1 gene silencing, and two of them (ALC1-si1 and
ALC1-si2) were able to effectively knock down the expression of ALC1 (Fig. 4A). Soft agar assay demonstrated
that the colony formation ability was significantly reduced (Student t test, P ⬍ 0.05) in cells in which ALC1
expression was silenced (Fig. 4B). Furthermore, DNA
content analysis by flow cytometry showed that ALC1-si1
was able to inhibit the cell cycle at the G1/S checkpoint
(Fig. 4C). The percentage of cells in the S phase was
Discussion
Like that of other solid tumors, the development of
HCC is a multiple-step process involving a sequence of
genetic changes, which includes amplification at 1q21.
Our previous study showed that amplification of 1q21 is
an early event in HCC development,14 implying that the
putative oncogene within this region may play an important role in the initiation of HCC pathogenesis. Here, we
report the identification and characterization of a novel
oncogene, ALC1, isolated from a 1q21 amplicon. Amplification and overexpression of ALC1 were detected in
over 50% of HCC cases. In the present study, the oncogenic role of ALC1 was supported by the following evidence: (1) ALC1-transfected cells were able to form more
colonies in soft agar and caused tumor formation in a
nude mouse, (2) the tumorigenicity of ALC1 could be
effectively inhibited by siRNA against ALC1, and (3)
ALC1 played an inhibiting role in apoptosis.
ALC1 belongs to the SNF2 superfamily, possessing
putative helicase sequence motifs similar to those
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Fig. 4. Silencing ALC1 expression by siRNA. (A) Two siRNAs (ALC1-si1 and ALC1-si2) could efficiently reduce the expression of ALC1 in H2-M cells.
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as an internal control. (B) Colony formation ability in soft agar was decreased
significantly in siRNA-treated H2-M cells (**P ⬍ 0.05). (C) Flow cytometry analysis showed that ALC1-si1 could inhibit the cell cycle at the G1/S
checkpoint. The percentage of cells in the S phase was decreased from 35% to 23.7%. (D) Western blot analyses indicated that p53 and p21Waf1/Cip1
were down-regulated, whereas cyclin E and Cdk2 were up-regulated in ALC1-transfected QGY-7703 cells in comparison with vector-transfected
QGY-7703 cells. ␤-Actin was used as a loading control. (E) Western blot results were quantified by densitometry, and data are presented as mean ⫾
standard error (n ⫽ 3). Fold values were first normalized with actin and then compared with vector-transfected QGY-7703 cells.
found in the proteins of helicase superfamily 2. SNF2
proteins can stabilize or perturb protein-DNA interactions by using the energy released by their DNAdependent ATPase activity and play important roles
in transcriptional regulation, maintenance of chromosome integrity, and DNA repair.15,16 Despite the
presence of helicase motifs, no protein in the SNF2
family has yet been shown to have helicase activity.17 A
comparison of the protein structures between CHD1
and ALC1 shows that both contain the SNF2_N domain and a helicase superfamily domain (HELICc).
CHD1 is able to bind DNA18 and regulate adenosine
triphosphate– dependent nucleosome assembly and
mobilization through their conserved double chromodomains and SNF2 helicase/ATPase domain.19 As a
result of its similarity to CHD1, ALC1 is also hypothesized to play important roles in transcriptional regulation, maintenance of chromosome integrity, and
DNA repair.
Promotion of cell proliferation is a major molecular
mechanism of an oncogene in cancer development. In
this study, we demonstrated that ALC1 could facilitate
DNA synthesis and promote G1/S phase transition in
ALC1-transfected cells. The cell proliferation role of
ALC1 can be effectively inhibited by siRNA against
ALC1. Further study showed that ALC1 could reduce
p53 expression. The p53 pathway is crucial for effective
tumor suppression in humans.20 The p53 protein is a
transcription factor that up-regulates the expression of
p21Waf1/Cip1, a Cdk inhibitor, responding to diverse
stresses (including DNA damage and overexpressed
oncogenes).21 p21Waf1/Cip1 serves as a key mediator in
G1/S transition through Cdk2 inhibition and regulation of the activity of cyclin E–Cdk2 complex, which
are essential for S phase entry.22,23 The reduced expression of p21Waf1/Cip1 facilitates the activation of cyclin
E–Cdk2 complex, which results in the cyclin E–Cdk2–
medicated retinoblastoma protein phosphorylation
and destruction of retinoblastoma protein–E2F binding. The releasing E2F activates the transcription of
genes necessary for S phase entry and progression.24 In
the present study, we demonstrated that overexpression of ALC1 could promote cell proliferation, at least
in part, via dysregulation of the p53–p21Waf1/Cip1– cy-
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509
regulated in ALC1-transfected QGY-7703 cells. Taken
together, our results suggest that ALC1 is the target
oncogene responsible for the 1q21 amplification event
and plays an important role in HCC pathogenesis via
the promotion of cell proliferation and the inhibition
of apoptosis.
References
Fig. 5. The inhibition role of ALC1 in apoptosis. (A) Representative
figures of TUNEL staining images. After cells were treated with STS for 4
hours, more apoptotic cells (bright white) were detected in vectortransfected QGY-7703 cells in comparison with ALC1-transfected 7703
cells. (B) Detection of the apoptotic index between ALC1-transfected and
vector-transfected QGY-7703 cells before and after STS treatment
(**P ⬍ 0.05). The data showed that ALC1-transfected QGY-7703 cells
could resist STS-induced apoptosis in comparison with QGY-7703 only.
(C) Expressions of caspase 3 and Bax were compared between ALC1transfected and vector-transfected QGY-7703 cells before and after STS
treatment by western blot analyses. ␤-Actin was used as a loading
control. (D) Protein levels of caspase 3 and Bax were quantified by
densitometry, and data are shown as mean ⫾ standard error (n ⫽ 3).
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that proapoptotic cytoplasmic Bax and caspase 3, the
executioner caspase of cellular apoptosis, were down-
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