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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References

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 2007.)

Hepatocellular 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 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 amplified 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

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References

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, QSG-7701, 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 μg 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 formalin-fixed 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 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), β-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 μM) for 4 hours. Morphological changes in the nuclear chromatin undergoing apoptosis were detected by TUNEL assay according to the manufacturer's protocol (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 ALC1-transfected 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

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References

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).

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Figure 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.

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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 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%) informative 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).

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Figure 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).

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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.

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Figure 3. Oncogenic ability of ALC1. (A) Expression of ALC1 in ALC1-transfected 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). ALC1-expressing 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.

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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 ALC1-transfected and blank vector–transfected LO2 cells, respectively (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 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 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).

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Figure 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. β-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.

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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).

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Figure 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 vector-transfected 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 ALC1-transfected and vector-transfected QGY-7703 cells before and after STS treatment by western blot analyses. β-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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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References

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 found in the proteins of helicase superfamily 2. SNF2 proteins can stabilize or perturb protein-DNA interactions by using the energy released by their DNA-dependent 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–cyclin E–Cdk2 pathway. Moreover, the effect of ALC1 on p53 and p21 regulation should be further clarified.

The decrease of apoptosis is another major mechanism of an oncogene in cancer development. ALC1 was able to decrease the apoptotic index in ALC1-transfected QGY-7703 cells in comparison with vector-transfected QGY-7703 cells. Further study showed that proapoptotic cytoplasmic Bax and caspase 3, the executioner caspase of cellular apoptosis, were down-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

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References