Abstract
OBJECTIVE Overexpression of growth factors and their receptors such as PDGF, FGF, VEGF, IGF, EGF, TGFα etc. play a critical role in the development and progression of malignant gliomas. AKT, one of the most potent downstream signaling effectors of these growth factor receptors is usually overactivated in malignant gliomas. The present study was undertaken to investigate the effects of antisense and dominant negative AKT2 RNA on the survival of glioma cells with overexpression of AKT2.
METHODS Antisense and dominant negative AKT2 constructs (AS-AKT2, DN-AKT2) were transfected into human glioblastoma cell line TJ905 with overexpression of AKT2. Using Western blotting, MTT assay, Ki67 labeling index (Ki67 LI), flow cytometry and the TUNEL method, the expression of AKT2 and GFAP, the proliferation rate and apoptosis of glioma cells transfected with AS-AKT2 or DN-AKT2 were compared to those characteristics of parental and glioma cells transfected with an empty vector.
RESULTS Cell proliferation was inhibited in glioma cells transfected with AS-AKT2 and DN-AKT2 RNA, while GFAP expression and apoptosis were markedly increased in those cells.
CONCLUSION AKT is an important mediator in the growth signaling pathway of malignant gliomas and is a potential promising therapeutic target for malignant gliomas.
keywords
AKT or PKB (protein kinase B) is a subfamily of the serine/threonine protein kinases that includes AKT1 (PKBα), AKT2 (PKBβ), and AKT3 (PKBγ). Each AKT family member contains an amino-terminal pleckstrin homology (PH), a kinase domain and a carboxyl-terminal regulatory domain. [1] AKT is activated by a variety of stimuli, such as growth factors and their receptor stimulation, stress, protein phosphatase inhibitors in a phosphotidylinositol 3’-OH kinase (PI3K)-dependent process. PI3K can phosphorylate phosphotidylinositol-3,4-bisphosphate (PIP2) to phosphotidylinositol-3,4,5-trisphosphate (PIP3). PIP3 binds to the PH domain of AKT, resulting in the phosphorylation on its 2 key residues, threonine T308 and serine S473, which are both necessary for AKT activation. The activity of AKT can be inhibited by the PTEN/MMAC1 tumor suppressor gene, SHIP and CTMP (carboxyl-terminal modulator protein). [2-4] There may be subtle differences in the AKT isoforms which remain to be identified. However, the oncogenic effects of the AKT isoforms are indistinguishable, [1,5] and AKT2 has been shown to be primarily involved in human malignancies.
It has been reported that AKT amplification and/or overexpression exist in a variety of human cancers such as ovarian, pancreatic, breast, prostate, thyroid, squamous cell carcinomas of the oral cavity and gliomas.[6-13] To further examine the role of AKT2 in mediating the development of glioma, antisense AKT2 (AS-AKT2) and dominant-negative AKT2 (DN-AKT2) expression plasmids were transfected into human malignant glioma cells to examine whether the malignant phenotype of glioma cells could be reversed. This would provide support for the therapeutic targets against gliomas.
Materials and Methods
Plasmid constructs
A plasmid carrying antisense AKT2 cDNA was constructed by inserting a 1.2 Kb EcoRI fragment from a human AKT2 cDNA clone into a LXSN retroviral vector in the antisense orientation. The fragment was generated by deleting 70 amino acids from the C-terminus of the open reading frame of AKT2. The dominant-negative AKT2 construct was obtained by inserting a 1.4Kb human AKT2 cDNA fragment mutated at E299K to the EcoRI site of the LSXN vector in the sense orientation.
Cell culture and transfection
The TJ905 glioblastoma cell line was established and characterized in our laboratory.[14] TJ905 cells (l× 105) were plated in 60 mm cell culture dishes and grown in DMEM supplemented with 10% fetal calf serum at 37 °C under 5% CO2 until they were 50-80% confluent. Expression plasmids AS-AKT2, DN-AKT2, and empty vector LXSN were transfected into TJ905 cells by lipofectamine (Life Technologies, USA). The LXSN vector has a neomycin resistant gene for selection of stable transfectants and positive stable clones were selected using G418 as previously described.[15] Three stable transfected clones for each construct were expanded for further studies.
Western blot analysis
Total soluble proteins from cultured glioma cells were extracted and the protein concentration determined by the Lowry method. Forty micrograms of protein lysates from each sample were subjected to SDS-PAGE on 10% acrylamide gel and the separated proteins transferred to a PVDF membrane. The blot was incubated with a primary antibody against AKT2 and GFAP (1:500 dilution), followed by HRP-conjugated secondary protein (1:1000 dilution). The specific protein was detected using a Super Signal protein detection kit (Pierce, USA). After washing with buffer, the PVDF membrane was rehybridized with a primary antibody for β-actin (1:500 dilution), followed by the same procedures as described above. The relative quantification was determined as the density of AKT2 divided by the density of β-actin.
MTT assay
The MTT assay was used for determination of the cell growth rate of transfected and non-transfected cells. Briefly, 4 × 103 cells were plated into each well of 96 well plates in triplicate. On each day for 6 consecutive days, 20 μl MTT (5 mg/ml) was added to each well, and the cells were incubated at 37 °C for an additional 4 h. Then the reaction was stopped by lysing the cells with 200 μl of DMSO for 5 min. Quantification (optical density) was measured at 570 nm and expressed as a percentage of control.
Ki67 immunohistochemistry
The proliferation of the cells was examined by the Ki67 labeling index (LI) using immunohistochemical methods. Cover slips plated with different groups of TJ905 glioma cells were fixed, washed and covered with 1:100 dilution of primary antibody for Ki67 (Santa Cruz, USA) overnight at 4°C, then biotinylated secondary antibody in a dilution of 1:200 was added at room temperature for 1 h, followed by incubation with ABC-peroxidase for an additional 1 h. After washing the cells with Tris-buffer, they were incubated with DAB (3, 3' diaminbenzidine, 30 mg dissolved in 100 ml Tris-buffer containing 0.03% H2O2) for 5 min, rinsed in water and counterstained with Hematoxylin. The Ki67 LI was determined as the fraction of positive staining cells in a total number of 500~ 1000 cells counted on each cover slip under a light microscope using high magnification.
Flow cytometry analysis
Non-transfected and transfected cells in the log phase of growth were harvested following trypsinization. Cells were incubated with RNase at 37°C for 30 min after which nuclei of the cells were stained with propidium iodide for an additional 30 min. A total of 10,000 nuclei were analyzed in a FACS Caliber flow cytometer (Becton Dickinson, USA) and the DNA histograms analyzed using Modifit software.
Detection of apoptosis
The extent of apoptotic cell death was detected by the TUNEL method using an in situ cell-death kit (Boehringer Mannheim, Germany) according to the supplier's instruction. The apoptotic index was calculated as the number of apoptotic cells per 500-1000 total cells counted using a light microscope under high magnification.
Statistical analysis
Data obtained in this study were tested for statistical significance by the one way Anova test and LSD using SSPS 10.0 version software. A P<0.05 was considered as a significant difference.
Results
AKT2 expression in transfected glioma cells
To examine the role of AKT2, TJ905 glioma cells were transfected with antisense AKT2 (AS-AKT2) and dominant-negative AKT2 (DN-AKT2) expression plasmids and stable clones were isolated. As shown by Western blotting, AKT2 protein expression was greatly suppressed in AS-AKT2 transfected cells, while their expression levels were not significantly changed in TJ905 glioma cells transfected with DN-AKT2 (Fig.1). This result confirms that antisense AKT2 expression can block the expression of endogenous AKT2 expression.
Western blot analysis of TJ905 cells transfected with DN-AKT2 and AS-AKT2. TJ905: TJ905 cells, LXSN: transfected with empty vector; DN1-3: three clones transfected with DN-AKT2; AS1-3: three clones transfected with AS-AKT2.
GFAP expression in transfected glioma cells
GFAP is the major intermediate filament of astrocytes and is closely associated with the growth and differentiation of astrocytes. It was demonstrated that GFAP expression was lowered in control glioma cells, while TJ905 cells transfected with AS-AKT2 and DN-AKT2 significantly induced the expression of GFAP (Fig.2A). Enhanced expression of GFAP indicates that the differentiation of malignant glioma cells may be induced by AS-AKT2 and DN-AKT2.
Effect of DN-AKT2 and AS-AKT2 on cell proliferation. (A)Westem blot analysis of GFAP expression of TJ905 cell lines transfected with AKT2 constructs; (B) Ki67 immunostaining of TJ905 cell lines transfected with AKT2 constructs; n=6; (C) MTT assay of different cell lines 1, 3 and 6 days after transfection.
Proliferation of transfected glioma cells
Ki67 immunohistochemistry was performed to examine the rate of cell proliferation, as determined by the Ki67 labeling index. The Ki67 labeling index was also markedly decreased in transfected cells compared to control cells. There was no statistical difference in proliferation activity between AS-AKT2 and DN-AKT2 transfected cells (Fig.2B). The MTT assay showed that AS-AKT2 or DN-AKT2 transfected TJ905 cells proliferated at significantly lower levels than the parental cells and the cells transfected with an empty vector (Fig.2C). These results suggest that the inhibition of AKT2 may reduce cell proliferation and induce differentiation.
Cell cycle analysis
Cell proliferation is closely associated with the modulation of cell cycle progression. From the results of flow cytometric analysis, it was shown that the percentage of the S phase fraction was decreased in the transfected cells, and the percentage of cells arrested in G0+G1 phase was increased (Fig.3). These results suggest that there is a change toward delaying cell cycle progression in glioma cells after transfection with AS-AKT2 and DN-AKT2.
Effect of DN-AKT2 and AS-AKT2 on cell cycle progression. (A) A representative dot plot of flow cytometry analysis; (B) Cell cycle analysis of TJ905 cells transfeceted with AKT2 constructs by flow cytometry analysis.
Apoptotic cell death
The extent of apoptotic cell death was examined by the TUNEL method, as shown in Fig.4A. There were nearly no apparent apoptotic cells found in the parental glioma cells or cells transfected with an empty vector. However, the number of apoptotic cells was prominently increased in glioma cells transfected with AS-AKT2 or DN-AKT2 (Fig.4B). This result demonstrates from the opposite perspective that AKT2 has an anti-apoptotic effect in tumor cells.
Effect of DN-AKT2 and AS-AKT2 on apoptotic cell death. (A) TUNEL staining of TJ905 cell lines transfected with AKT2 constructs; (B) Measurement of apoptosis index, n=6.
DISCUSSION
In the present study, we have demonstrated that AKT2 expression was significantly upregulated in the TJ905 glioma cell line. Following transfection of AS-AKT2 and DN-AKT2 into TJ905 cells, GFAP expression was upregulated, the cell proliferation rate was reduced, and apoptotic cell death was induced. These observations provide strong evidence that regulation of AKT2 expression and activity may be important in controlling the development and malignant progression of gliomas.
Mounting evidence demonstrates that many growth factors and their receptors are either overexpressed or overactivated in gliomas, including FGF, EGF, TGFα, VEGF and IGF. Alteration of EGFR has been reported to be an early and frequent molecular event of the most malignant glioma-glioblastoma multiforme (GBM).[16-18] These growth factor receptors belong to the receptor tyrosine kinase (RTK) family. One of the RTK-mediated signaling pathways that plays a pivotal role in the tumorigenesis of gliomas is PI3K/AKT. AKT activation is negatively regulated by the tumor suppressor gene PTEN, which has been shown to be non-functional in 20~44% of glioblastomas. [19,20] Therefore, AKT is overexpressed in many, if not all, gliomas due to EGFR overepression and the loss of PTEN function. AKT has pleiotropic effects implicated in tumorigenesis. It exerts anti-apoptotic activity by inactivating the pro-apoptotic member of the Bcl-2 family of proteins, Bad, and the Forkhead transcription factor, procaspase-9. AKT can also induce NF-κB-dependent transcription of anti-apoptotic genes. AKT enhances telomerase activity through phosphorylation of human telomerase reverse transcriptase (hTERT). AKT deregulates cell growth by inhibition of p21, p27 and stabilization of cyclin D and promotes nuclear entry of mdm2, leading to inactivation of the p53 pathway that may also contribute to uncontrolled cell proliferation. It also enhances cell invasiveness and angiogenesis by stimulating secretion of MMPs and activation of endothelial nitric oxide synthase (eNOS).[1,5,21-23]
There are some reports concerning the alteration of the PI3K/AKT signaling pathway in gliomas. Their results showed that AKT was activated in malignant gliomas and closely correlated with EGFR overexpression.[12,13] The use of the antisense AKT2 approach against malignant glioma cells has not been reported. However, the results in the present study are similar to that reported by Cheng et al.[8] in that the growth, invasiveness and tumorigenecity were greatly inhibited after transfection with antisense AKT2 in human pancreatic cancer cells with highly elevated levels of AKT2. Furthermore, Narita et al. [24] also demonstrated that transfection of a kinase dead dominant negative AKT mutant into UM87 glioma cells with mutant EGFR (EGFR vIII) expression resulted in down-regulation of tumorigenecity and up-regulation of the cyclin-dependent kinase inhibitor p27 in vivo.
In conclusion, AKTs are important downstream signaling proteins of growth factors and growth factor receptors which are usually overexpressed in human gliomas. The data of the current study further demonstrate that inhibition of AKT2 expression by AS-AKT2 and DN-AKT2 could reverse the malignant phenotype of glioma cells. AKT2 can be developed as a therapeutic target for malignant gliomas
Footnotes
This work was supported by the National Natural Science Foundation of China (No. 30100050).
- Received January 29, 2005.
- Accepted April 22, 2005.
- Copyright © 2005 by Tianjin Medical University Cancer Institute & Hospital and Springer
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