In normal cells, proliferation is tightly regulated by multiple intrinsic and extrinsic signaling pathways. Cells become cancerous when they acquire genetic alterations that allow them to evade such regulatory mechanisms. The function of tumor suppressor genes is to implement such regulation, and these genes are often inactivated in cancer cells.
ING (inhibitor of growth) genes are a novel tumor suppressor gene family, and the known members of human ING family (ING1, ING2, ING3, ING4, and ING5) are implicated in a variety of processes including gene transcription, oncogenesis, apoptosis, DNA repair, and cell cycle control (Nagashima et al.,2001,2003; Feng et al.,2002; Shiseki et al.,2003; Campos et al.,2004). ING4, a novel member of the ING family, has recently emerged as a strong tumor suppressor gene that functions in cell proliferation, contact inhibition, and angiogenesis (Garkavtsev et al.,2004; Kim et al.,2004). ING4, along with ING5, was first isolated and characterized through a computational search of ING1 sequence homology (Shiseki et al.,2003). It maps to human chromosome 12p13-31, including 8 exons and 7 introns, and encodes a 248 amino acid polypeptide. ING4 is a nuclear factor expressed in normal human tissues, but its expression is markedly reduced in astrocytic neoplasms, such as human gliomas, with levels inversely correlated with the tumor grade (Garkavtsev et al.,2004). Previous studies demonstrated that overexpression of ING4 could induce a decreased cell population in the S phase of the cell cycle and apoptosis in a p53-dependent manner with increasing p21 expression in RKO cells (Shiseki et al.,2003). ING4 could also suppress the loss of contact inhibition elicited by MYCN or MYC (Kim et al.,2004), induce significant G2/M arrest of cell cycle, and enhance chemosensitivity to doxorubicin and etoposide in HepG2 cells (Zhang et al.,2004). Finally, ING4 could inhibit NF-kB activation by physically interacting with the p65 (RelA) subunit of nuclear factor NF-kB, resulting in transcriptional repression of the downstream NF-kB–responsive genes (such as IL-8, IL-6, COX-2, and CSF-3) and many angiogenesis-related genes, causing further inhibition of tumor angiogenesis (Garkavtsev et al.,2004).
Adenocarcinoma, the most common form of lung cancer, is one of main human malignant tumors, and its occurrence and development are highly correlated with inactivation of tumor suppressor genes. ING4, as a novel candidate tumor suppressor gene, has been implicated in several human malignancies such as gliomas, breast tumors, and squamous cell carcinomas (Garkavtsev et al.,2004; Kim et al.,2004; Gunduz et al.,2005). It also plays a role in negatively regulating tumor growth and apoptosis. To confirm the inhibitory tumor growth effects of ING4 on lung adenocarcinoma, we cloned the full-length coding sequence of the human ING4 gene and transfected it into human lung adenocarcinoma A549 cells to investigate the effects of ING4 on A549 cell proliferation. The mechanism of how ING4 negatively regulates tumor growth remains largely unknown. Thus, we also examined the inhibitory mechanism of ING4 on tumor growth of lung adenocarcinoma.
The Wnt signaling pathway is important during carcinogenesis. Wnt-1/β-catenin–mediated transcriptional activation has been implicated in several human malignancies such as breast cancer and prostate carcinoma (Wong et al.,2002; Chen et al.,2004). Therefore, we also tested the effect of ING4 overexpression on activation of the Wnt-1/β-catenin signaling pathway and the expression of its target proteins p27, cyclinD1, SKP2, and Cox2 in A549 cells. Furthermore, we assessed the effects of ING4 on the sensitivity of A549 cells by radiotherapy and chemotherapy.
MATERIALS AND METHODS
Materials and Cell Culture
The Therm RT-PCR/plat Taq, QIAGEN Plasmid Maxi Kit, QIAqick Gel Extraction Kit, Lipofectamine 2000, and G-418 were purchased from Invitrogen Corporation (Carlsbad, CA); the goat polyclonal antibody against ING4 (ab3714) from Abcam Inc. (Cambridge, MA); and the goat polyclonal antibody against Wnt-1, mouse monoclonal antibodies against β-catenin, p27, Cox2, and GAPDH, and rabbit monoclonal antibodies against SKP2 and cyclinD1 from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). The human lung adenocarcinoma cell line A549 was purchased from the Department of Immunology of Harbin Medical University. Cells were grown in DMEM medium supplemented with 10% fetal bovine serum (Gibco Inc., Carlsbad, CA) at 37°C with 5% CO2.
Detection of ING4 mRNA in Human Lung Adenocarcinoma Tissue
In vitro, the transcription of ING4 mRNA was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) in normal lung tissues and in various grades of lung adenocarcinoma tissues conserved in liquid nitrogen. Total RNAs were isolated with Trizol reagent from 10 cases of normal lung tissues, 10 cases of well-differentiated lung adenocarcinoma tissues, and 10 cases of poorly differentiated lung adenocarcinoma tissues. cDNA was synthesized and amplified by RT-PCR. RT-PCR was performed using a first-strand synthesis kit and a PCR kit with ING4 upstream and downstream primers, and β-actin as a control. The primers for the human ING4 gene were designed with reference to (GeneID: 51147). The EcoRI site was inserted into the forward primer, and the XBaI site was into the reverse primer. The primer sequences were as follows: the forward primer p1: 5′-TTG AAT TCA TGG CTG CTG GGA TGT ATT TGG GAAC-3′; the reverse primer p2: 5′-CCT CTA GAT CAG ATA CAT CCA CAC CTT TTA GCG-3′. The primer sequences of β-actin were as follows: the forward primer p1: 5′-ACCACAGTCCATGCCATCAC-3′; the reverse primer p2: 5′-TCCACCACCC TGTTG CTGTA-3′.
Construction of Recombinant Plasmid pcDNA3.1-ING4 and Transfection
Total RNA was extracted from normal human gastric mucosa with Trizol reagent. A cDNA fragment encoding ING4 was synthesized and amplified by RT-PCR. The ING4 cDNA fragment was subcloned into a pcDNA3.1 plasmid vector by EcoRl/XBal double-digestion and designated as pcDNA3.1-ING4. The recombinant plasmid was amplified in Escherichia coli DH5α, and was identified by PCR and DNA sequencing. Then pcDNA3.1-ING4 was transfected into A549 cells with Lipofectamine 2000 reagent. Empty vector pcDNA3.1 was used as a negative control. The colonies were selected in the medium containing G418 (600 μg/ml) for 2 weeks. G418-resistant colonies were identified at the gene and protein levels by RT-PCR and Western blot analysis, respectively.
Cell Proliferation Analysis
To observe cell proliferation, the transfected A549 cells were seeded in 96-well plates at a density of 1 × 103 cells/well and cultured for 24 hr, and the cell viability was measured by MTT assay. Simultaneously, a colony formation assay was used to examine cell proliferation. Cell suspensions with 1,000 exponential transfected A549 cells were inoculated on flat plates (diameter 60 mm), cultured for 2 weeks, then the number of the colonies was counted. To evaluate the effects of ING4 on the sensitivity of A549 cells to some DNA-damage agents, A549 cells with or without exogenous ING4 gene were seeded in 96-well plates at a density of 1 × 103 cells/well, cultured for 24 hr, and subsequently treated with 5-Fu (6.25 μmol/L). Three days after the treatment stopped, the cell viability was observed by MTT assay. At the same time, a cell suspension with 1,000 exponential A549 cells with or without exogenous ING4 gene were inoculated on flat plate and cultured for 24 hr, and subsequently exposed to γ-irradiation (up to 60 Gy; 20 Gy/dx3), and the cell proliferation was measured by colony formation assay. All experiments were performed in triplicate at minimum.
Western Blot Analysis
A549 cells with or without exogenous ING4 gene were harvested with lysis buffer, total proteins were extracted, and the protein concentration in the lysate was determined using a spectrophotometer. Lysate containing 70 μg of protein was loaded on to 12% acrylamide gels, subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA), and subsequently transferred onto a polyvinylidene difluoride membrane by electroblotting. Immune complexes were visualized with specific antibodies against ING4 (dilution; 1:500) and cyclinD1, p27, SKP2, Cox2, Wnt-1, and β-catenin (dilution; 1:200). The secondary antibodies used were mouse anti-goat, mouse anti-rabbit, and rabbit anti-mouse IgG.
Tumorigenicity of Transfected A549 Cells in Nude Mice
Forty BALB/C (nu/nu) male nude mice (16–20 g; 4–6 weeks of age) were purchased from the Animal Institute at the Chinese Academy of Medical Sciences. The animals were fed in a pathogen-free environment with constant temperature and humidity, and allowed standard rat chow and water ad libitum. They were maintained on a 12-hr light–dark schedule. All the experimental procedures were reviewed and approved by the Animal Care and Use Committee of Harbin Medical University. Nude mice were randomly divided into four groups, and the tumorigenic experiments in vivo were repeated twice with five mice per group. Exponential A549 cells with exogenous ING4 gene at a density of 1 × 107 cells/ml were subcutaneously inoculated with 0.2 ml into the abdomen of nude mice. A549 cells with empty vector and nontransfected A549 cells were used as the control groups, and physiological saline was the blank group. All the animals were killed by transection of the spinal cord 4 weeks later, and these mice were weighed and the xenografts were measured in weight and volume. The tumor growth was evaluated by the tumor volume calculated by the formula V = ab2/2, where a represented the longest diameter and b the diameter perpendicular to a.
Data were expressed as mean ± standard deviation. SPSS statistic soft package was used to perform one-way analysis of variance, q-test. P < 0.05 was considered statistically significant.
Expression of ING4 Gene in Various Grades of Lung Adenocarcinoma Tissues
RT-PCR analysis revealed that the transcription level of ING4 mRNA in lung adenocarcinoma tissues was significantly reduced as compared with normal lung tissues (P < 0.01; Fig. 1). Furthermore, the expression level in poorly differentiated lung adenocarcinoma tissues was lower than that in well-differentiated lung adenocarcinoma tissues (P < 0.01; Table 1).
Table 1. Density value analysis of band brightness for ING4/β-actin (x ± SD, n = 10)a
No. of cases
Ratio for ING4/β-actin
The level of ING4 in normal lung tissues was significantly higher than that in lung adenocarcinoma tissues (q = 27.45, 43.96; P < 0.01), and the level of ING4 in well-differentiated lung adenocarcinoma was significantly higher than that in poorly differentiated lung adenocarcinoma (q = 16.51; P < 0.01).
Normal lung tissues
1.434 ± 0.133
Well-differentiated lung adenocarcinoma
0.719 ± 0.037
Poorly differentiated lung adenocarcinoma
0.289 ± 0.036
Cloning of ING4 cDNA and Identification Analysis of the Recombinant Plasmid pcDNA3.1-ING4
The target gene fragment was isolated and amplified by RT-PCR from normal human gastric mucosa, after which the recombinant pcDNA3.1-ING4 was constructed. RT-PCR analysis showed that a single fragment was visualized as a band of approximately 750 bp on a 1% agarose gel (Fig. 2). DNA sequencing confirmed that the recombinant plasmid contained the ING4 gene fragment of the expected size, which agreed with the sequence of the ING4 gene confirmed from GenBank.
Expression of ING4 in A549 Clones With or Without ING4 Gene
RT-PCR analysis showed that the level of ING4 mRNA in A549 clones with pcDNA3.1-ING4 was significantly higher than that in A549 clones with pcDNA3.1 (Fig. 3). The level of the target gene product by Western blot analysis revealed a significant difference in A549 clones with exogenous ING4 gene as compared with A549 clones with empty vector and nontransfected A549 cells (Fig. 4).
ING4 Negatively Regulates A549 Cell Growth In Vitro and In Vivo
MTT assay revealed that the OD value of A549 clones with pcDNA3.1-ING4 was lower than that of A549 clones with pcDNA3.1 (Fig. 5; Table 2; t = 16.81; P < 0.01), suggesting that the cell viability of A549 clones with pcDNA3.1-ING4 was significantly decreased. Similar results were obtained by cell colony formation assay, as the number of colonies in A549 clones with pcDNA3.1-ING4 was less than that of A549 clones with pcDNA3.1 (Fig. 6; Table 2; t = 53.16; P < 0.01). These data suggest that the overexpression of ING4 might negatively regulate A549 cell growth in vitro.
Table 2. MTT and the colony formation assay of A549 clones with and without exogenous ING4 gene (x ± SD, n = 6)a
No. of samples
OD value in MTT test
No. of colonies
MTT OD value after treated with 5-FU
No. of colonies after treated with γ-ray
A significant difference was observed between A549 clones with pcDNA3.1-ING4 and A549 clones with pcDNA3.1 in all of the above tests (P < 0.01).
A549 clones with pcDNA3.1-ING4
0.628 ± 0.239
54.800 ± 11.063
0.156 ± 0.014
19.000 ± 4.289
A549 clones with pcDNA3.1
0.957 ± 0.032
112.000 ± 9.389
0.190 ± 0.022
54.000 ± 5.865
In vivo, an effect of ING4 on growth of A549 cells was observed in nude mice. Four weeks after inoculation, the xenograft size in the group inoculated with A549 clones with pcDNA3.1-ING4 was significantly smaller than that in the control group (Fig. 7). In the group of A549 clones with pcDNA3.1-ING4, the tumor growth was significantly inhibited and the tumor volume and weight were significantly decreased as compared with the control groups (P < 0.01 and P < 0.05; Table 3).
Table 3. Repressive effect of ING4 on xenografts growth of nude mice (x ± SD, n = 5)a
No. of samples
Weight of nude mice (g)
Weight of xenografts (g)
Volume of xenografts (mm3)
A statistically significant difference was observed in the weight of nude mice and the volume and weight of xenografts between A549 clones with pcDNA3.1-ING4 group and A549 clones with pcDNA3.1 group or nontransfected A549 clones group. In the weight of nude mice: q = 9.56, P < 0.01 for A549 clones with pcDNA3.1-ING4 and A549 clones with pcDNA3.1; q = 12.34, P < 0.01 for A549 clones with pcDNA3.1-ING4 and nontransfected A549 clones. In the weight of xenografts: q = 4.62, P < 0.05 for A549 clones with pcDNA3.1-ING4 and A549 clones with pcDNA3.1; q = 6.17, P < 0.01 for A549 clones with pcDNA3.1-ING4 and nontransfected A549 clones. In the volume of xenografts: q = 9.74, P < 0.01 for A549 clones with pcDNA3.1-ING4 and A549 clones with pcDNA3.1; q = 10.82, P < 0.01 for A549 clones with pcDNA3.1-ING4 and nontransfected A549 clones. No significant difference was observed in the weight of nude mice and the volume and weight of xenografts between A549 clones with pcDNA3.1 group and nontransfected A549 clones group: q = 2.78, 154, 1.07; P > 0.05.
A549 clones with pcDNA3.1-ING4
23.11 ± 0.47
0.75 ± 0.04
551.57 ± 25.36
A549 clones with pcDNA3.
25.28 ± 0.52
0.87 ± 0.06
747.56 ± 52.56
1Nontransfected A549 clones
25.91 ± 0.31
0.91 ± 0.07
Molecular Mechanism of Negative Regulation of A549 Cell Growth by ING4
To determine the molecular mechanism involved in the inhibitory effect of ING4 on A549 cell growth, we first examined the effects of ING4 on the level of the cell proliferation-regulating proteins cyclinD1, p27, SKP2, and Cox2 by Western blot analysis. Results showed that, when compared with A549 clones with empty vector, the level of p27 was significantly up-regulated and the level of cyclinD1, SKP2, and Cox2 was down-regulated in A549 clones with pcDNA3.1-ING4 (Fig. 8).
Subsequently, we explored the mechanisms involved in the effects of ING4 on the above proteins by examining the effects of ING4 overexpression on the level of Wnt-1 and β-catenin in A549 cells by Western blot analysis. The levels of Wnt-1 and β-catenin were decreased in A549 clones with pcDNA3.1-ING4 (Fig. 8). These data indicate that ING4 might negatively regulate A549 cell growth by means of inducing inactivation of the Wnt-1/β-catenin pathway, and further influencing the expression of cell proliferation-regulating proteins such as cyclinD1, p27, SKP2, and Cox2.
ING4 Enhances the Sensitivity of A549 Cells to Radiotherapy and Chemotherapy
A549 clones, with or without exogenous ING4 gene, were treated with 5-Fu (6.25 μmol/L), and the OD value of the wells subsequently determined at 490 nm by MTT method. Results showed that the OD value of A549 clones with pcDNA3.1-ING4 was reduced as compared with A549 clones with pcDNA3.1 (Fig. 9; Table 2; t = 16.19; P < 0.01). At the same time, A549 clones, with or without exogenous ING4 gene, were irradiated with γ-rays (up to 60 Gy), and the cell growth subsequently measured by colony formation assay. Results revealed that the number of colonies was reduced in A549 clones with pcDNA3.1-ING4 as compared with A549 clones with pcDNA3.1 (Fig. 10; Table 2; t = 64.62; P < 0.01).
ING4, a new candidate tumor suppressor gene, has been implicated in negatively regulating cell proliferation, inducing apoptosis, and angiogenesis; however, the precise mode of action for ING4 has yet to be elucidated. It is clear that ING4 has a markedly low-expression in many human malignant tumors, such as human gliomas, with the levels inversely correlated with the tumor grades (Garkavtsev et al.,2004), or shows a deletion such as in human breast cancer and squamous cell carcinomas (Kim et al.,2004; Gunduz et al.,2005). In the present study, we showed that the expression of ING4 was markedly reduced in lung adenocarcinoma by RT-PCR as compared with normal lung tissues. Furthermore, the expression level in poorly differentiated lung adenocarcinoma was lower than that in well-differentiated lung adenocarcinoma. As a tumor suppressor gene, these data suggest that ING4 might be involved in the occurrence and development of lung adenocarcinoma.
Previous studies have demonstrated that ING4 plays a role in regulating brain tumor growth and angiogenesis (Garkavtsev et al.,2004). To further confirm the effects of ING4, we investigated the tumor growth inhibition of ING4 on lung adenocarcinoma and its mechanism by ING4 cDNA transduction into the human lung adenocarcinoma A549 cells. We first examined the effects of ING4 overexpression on the proliferation of A549 cells. In vitro, MTT assay indicated that A549 clones with exogenous ING4 gene exhibited a remarkably decreased cell viability, while the number of colonies of A549 clones with exogenous ING4 gene was significantly reduced by colony formation assay. Furthermore, in nude mice in vivo the tumorigenicity of A549 clones with constitutive overexpression of ING4 was significantly decreased as compared with A549 clones with empty vector and nontransfected A549 cells. These data suggest that the constitutive overexpression of ING4 might indeed play an inhibitory role on the proliferation of A549 cells and tumor growth in vitro and in vivo.
The mechanism by which ING4 negatively regulates tumor growth remains largely unknown. To determine molecular basis for the inhibitory effect of ING4 overexpression on A549 cell proliferation, we analyzed the effects of ING4 on the expression of cell proliferation-regulating proteins cyclinD1, p27, SKP2, and Cox2. CyclinD1, a known key regulator of cell proliferation, plays a critical role in cell cycle progression during mid G1 by initiating the multistep process that leads to pRb inactivation (Weinberg,1995). p27, an important cdk inhibitor, acts as a key negative regulator of the cyclin-cdk complex activity, and prevents cell cycle progression. It appears to be both necessary and sufficient to arrest cells before the late G1 restriction point (Coats et al.,1996). SKP2 can specifically recognize p27 with a replaced Thr187 and induces p27 degeneration by ubiquitination and cell proliferation through the S phase from the G1 phase. Skp2-dependent degradation of p27 is essential for cell cycle progression. In the present study, constitutive overexpression of ING4 significantly up-regulated the expression of p27 and down-regulated the expression of cyclinD1 and SKP2, suggesting that ING4 might inhibit A549 cell proliferation by arresting cell cycle progression in late G1 before pRb hyperphosphorylation. Cox2, a central enzyme in the prostaglandin biosynthetic pathway, plays a key role in the early stages of carcinogenesis by promoting tumor cell proliferation and their resistance to apoptosis, as well as angiogenesis, tumor cell invasion, and setting up of the metastatic process (Möbius et al.,2005; Tzankov et al.,2007). We found that constitutive overexpression of ING4 decreased the expression of Cox2, which might therefore be a pathway through which ING4 exerts its inhibitory action on A549 cell proliferation and tumor growth.
ING4 can regulate negatively A549 cell proliferation by up-regulation or down-regulation of cell proliferation-regulating proteins such as p27, cyclinD1, SKP2, and Cox2. This finding led us to investigate the mechanism involved in the modulation process of ING4 on these proteins. The Wnt signaling pathway is important during carcinogenesis. Activation of Wnt signaling through β-catenin/TCF (T-cell factor) complexes is a key event in the development of various tumors. Wnt-1 appears to signal by means of a unique pathway, thought to be initiated by interaction of Wnt-1 with its transmembrane receptors, leading to stabilization of a cytosolic pool of β-catenin. Accumulated β-catenin can translocate to the nucleus, interact with TCF transcription factors, and thereby mediate transcriptional activation. Wnt-1/β-catenin–mediated transcriptional activation has been implicated in several human malignancies (Wong et al.,2002; Chen et al.,2004). Multiple key oncogenic proteins, including c-Myc, cyclinD1, and Cox-2, have been found to be regulated by the Wnt-1/β-catenin signaling pathway. We speculated that ING4 might play an inhibitory function on cell proliferation and tumor growth of lung adenocarcinoma by means of Wnt-1/β-catenin signaling, and analyzed the expression of Wnt-1 and β-catenin in A549 clones with constitutive overexpression of ING4. A significant down-regulation of Wnt-1 and β-catenin was observed in A549 clones with constitutive overexpression of ING4. These data suggest that ING4 might induce inactivation of the Wnt-1/β-catenin signaling pathway by decreasing cytosolic accumulation of β-catenin by means of down-regulation of Wnt-1. CyclinD1, p27, and Cox-2 all serve as targets modulated by Wnt-1/β-catenin signaling pathway activation (Howe et al.,1999; Castro et al.,2001; Castelo-Branco et al.,2003; Hulit et al.,2006), of which the down-regulation of p27 and the up-regulation of cyclinD1 and Cox-2 were in response to Wnt-1/β-catenin signaling activation in tumor cells. In the present study, we found up-regulation of p27 and down-regulation of cyclinD1, SKP2, and Cox-2 in A549 clones with constitutive overexpression of ING4, which presented with a low-expression of Wnt-1 and β-catenin. Thus, overexpression of ING4 might up-regulate the expression of p27 and down-regulate the expression of cyclinD1, SKP2, and Cox2 by inducing inactivation of the Wnt-1/β-catenin signaling pathway.
ING4 has been reported to enhance cell death triggered by DNA-damage agents and to enhance the chemosensitivity to doxorubicin and etoposide in HepG2 cells (Zhang et al.,2004). To further confirm whether ING4 could affect the efficacy of radiotherapy or chemotherapy on lung adenocarcinoma, we evaluated the potential effects of ING4 on A549 cells irradiated with γ-rays or treated with the chemotherapeutic drug 5-Fu. The viability of cells in both groups treated with 5-Fu was markedly reduced as compared with the corresponding nontreated cells, while the viability of A549 clones with exogenous ING4 gene was further reduced compared with A549 clones with empty vector. Similarly, when irradiated with γ-rays, the cell growth of A549 clones with exogenous ING4 gene as examined by colony formation assay was decreased as compared with the control group, while the number of colonies of the two groups irradiated with γ-rays was lower than the corresponding nonradiated cells. These groups of data suggest that ING4 enhances the sensitivity of A549 cells to both radiotherapy and chemotherapy.
In conclusion, the present study indicates that the expression of ING4 was markedly reduced in human lung adenocarcinoma. ING4, as a tumor suppressor gene, negatively regulated A549 cells proliferation and tumor growth by up-regulation or down-regulation of cell proliferation-regulating proteins such as p27, cyclinD1, SKP2, and Cox2, by means of inactivation of Wnt-1/β-catenin signaling. Of interest, ING4 could also enhance the radiosensitivity to γ-rays and the chemotherapeutic sensitivity to 5-Fu in A549 cells, suggesting that ING4 may be a candidate for gene therapy, suggesting a new approach for treating patients with lung adenocarcinoma in combination with radiotherapy or chemotherapy.
We thank Xiaoyi Huang (from the Department of Genetics in Harbin Medical University of China) for his technical support and providing some material for this subject, and Yue Yang (from the affiliated Tumor Hospital of Harbin Medical University) for her assistance with the animal experiments.