Introduction

Apoptosis is an active form of programmed cell death different from necrosis. Like cell proliferation and differentiation, apoptosis is a characteristic event or process which is regulated by both intracellular and extracellular signals1, 2, 3. Recently, the increasing evidence implies that alteration in apoptosis contribute to tumorogenesis and thus treatment designed to alter apoptosis may have the potential to change the progression of cancer4.

The epidermal growth factor receptor (EGFR) is a 170 KD glycoprotein which contains essentially an extracellular binding domain, a transmembrane domain and an intracellular protein kinase domain. The EGFR tyrosine kinase-mediated signal transduction pathways have been considered to regulate many cellular processes. Amplification and overexpression of the EGFR has been detected in a number of human tumors of epithelial origin5. The sequence homology has been found between intracelluar portion of the EGFR and gp68v-erbB, the transforming protein of oncogene v-erbB 6. The EGFR activation has been implicated in autocrine stimulation of cell growth in many experimental system7. Therefore, the EGFR might be responsible for the genesis and the maintenance of malignant properties of certain types of tumors and it appears to be a good target for antitumor therapy.

In the previous studies, we have demonstrated the EGFR gene expression and mitogenic effects of EGF in cells of a human hepatoma cell line BEL-74048. Furthermore, we have obtained an antisense EGFR vector transfected BEL-7404 cell clone, termed JX-1 cells, showing amazing alteration in growth behaviors9. We describe here that antisense EGFR sequence enhances apoptotic cell death in the transfected JX-1 cells and its underlying mechanism will be discussed.

Materials and Methods

Materials

Fluo-3/AM, propidium iodide and thapsigargin were purchased from Sigma (St. Louis, MO, USA). EGF was the product from Gibco BRL (Gaithersburg, MD, USA).

Cell culture

Human hepatoma cell line BEL-7404 was established in our Institute. BEL-7404 cells and the derivatives were grown in Dulbocco's modified Eagles's medium (DMEM, Gibco, Grand Island, NY, USA) supplemented with 13% fetal calf serum, 100 u/ml penicillin and 100 mg/ml streptomycin in 5% CO2 incubator at 37 °C and cultured medium was changed every 48 h.

Stable transfectants

The recombinant plasmid containing antisense EGFR sequence was co-transferred with pSV2-neo into BEL-7404 cells by electroporation, followed by selection and cloning in the presence of G418 (400 μg/ml). The parent vector plasmid pEV-1 was used as control for transfection. Two representative cell lines JX-1 transfected with antisense EGFR sequence and JX-0 transfected with control vector pEV-1 were cloned for further analysis.

Electron microscopy

For transmission electron microscopy analysis, exponentially growing JX-1 cell in monolayer culture were collected by trypsinizing. The cells were fixed in 4% glutaraldehyde, postfixed in 1% OsO4, and embedded with Epon618. Ultrathin sections were cut and stained with uranyl acetate and lead citrate and viewed in a JEWl00B electron microscope.

Flow cytometry

The cells grown in monolayer culture were trypsinized and centrifuged at 250×g for 5 min. The cell pellet was resnspended in 1 ml hypotonic fluorochrome solution (propidium iodide 50μg/ml in 0.1% sodium citrate plus 0.1% Triton X-100, Sigma). The sample was kept at 4 °C in dark overnight. The propidium iodide fluorescence of individual cells was measured by using a FACStar Plus flow cytometry (Becton and Dickson). The cells with subdiploid DNA content were considered as apoptotic cells. Each value represented the mean of 10,000 cells.

DNA fragmentation Elisa assay

Cellular DNA fragmentation was measured by using a commercial available DNA fragmentation Elisa kit (Boehringer Mannheim) which is based on the quantitative sandwisch enzyme immunoassay principle using two mouse monoclonal antibodies directed against DNA and BrdU respectively. This allows the specific detection and quantification of Brdu-labeled DNA fragments. Briefly, the cells (1×104) were seeded to 96-weU flat bottom cell culture plate (Corning). Exponentially growing cells were incubated with 10μM BrdU for 12 h in 5% CO2 incubator at 37 °C and then lysed by adding lysis buffer. After lysis, the samples were centrifuged at 250×g for 10 min and 100 μ1 of supernatant removed from each sample for Elisa assay. The samples were added to microtiter plate precoated with anti-DNA antibody and incubated for 90 min. After the samples were incubated with anti-BrdU peroxidase for another 90 min, TMB substrate was added to plate. After incubation in the dark for 20 min, the reaction was terminated by adding 25μl of 1M H2SO4 to each well. Measurement of absorbance at 450 nm was performed using microplate reader (Bio-Rad).

Measurement of intracellular free Ca2+ concentration

Cytosolic Ca2+ concentration was measured by fluorometry of furo-2-1oaded or fluo-3-1oaded cells as described18.

Results and Discussion

EGF/EGFR pathway is known as an important regulating network for the growth control in either normal cells or malignant cells. We reported here the study of the role of EGFR in apoptosis of human hapatoma cells using antisense EGFR vector transfectants as a model. Flow cytometry combined with propidium iodide-staining showed that an increased number of sub-G1 phase cells, which contained subdiploid DNA, occurred in antisense EGFR transfected cell line JX-1 cells in comparison to those in control vector transfectants JX-0 cells and the parent BEL-7404 cells (Tab 1, left column). The percentage of cells with subdiploid DNA content in JX-1 cells was 9.5%, whereas those in JX-0 and BEL-7404 cells were similar and about 1.7%, indicating an enhanced rate of spontaneous apoptotic cell death in JX-1 cells. In the previous study9, we have demonstrated that BEL-7404 and control vector transfectants JX-0 cells can grow in serum-free medium, while JX-1 cells can not survive during the serum-starvation. In the present study, it was found that after serumstarvation for 24 h, the rate of apoptosis of JX-1 cells was increased up to 18.4%, but JX-0 and BEL-7404 cells only showed less than 2-3% apoptotic cells (Tab 1, right column). When the serum-starvation was elongated to 72 h, as shown in Fig 1, the rate of apoptotic cells with subdiploid DNA content were increased up to 33.7% in JX-1 cells, but less than 5% apoptotic cells were found in JX-0 and BEL-7404 cells. Data obtained with DNA fragmentation Elisa test also supported that JX-1 cells had a higher rate of apoptotic cell death than the control vector transfectants, especially during serum-starvation (Tab 2). Ultrastructure analysis of JX-1 cells demonstrated the occurrence of some characteristic morphologic changes of apoptosis, including condensation and fragmentation of chromatin and formation of apoptotic bodies (Fig 2).

Tab 1 The effects of serum starvation on apoptosis of BEL-7404, JX-0 and JX-1 cells*
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Fig 1
figure 1

Flow cytometric DNA content analysis of BEL-7404, JX-0 and JX-1 cells. These cells were grown in the absence of serum for 72 h before harvesting. X-axis represents fluorescence intensity and Y-axis represents cell members.

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Tab 2 Effects of serum-deprivation on DNA fragmentation in JX-0 and JX-1 cells*
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Fig 2
figure 2

Ultrastructure of JX-1 cells in the absence of serum for 48 h, observed by transmission electron microscope (x 6,000)

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Effects of EGFR on apoptosis is steadily gaining attention in recent years10, 11, 12, 13. EGF can maintain the survival of the mouse embryo cell under serum-free condition, while deprivation of EGF induces these cells undergoing apoptosis10. An anti-EGFR monoclonal antibody has been reported to block activation of EGFR tyrosine kinase and induce apoptosis in some human colorectai carcinoma cells11. We also observed that the monoclonal antibody against EGFR obviously inhibited the growth and induced the apoptosis of BEL-7404 cells in vitro (unpublished data). However, Armstrong et al. have found that activation of EGFR induces apoptosis in some human breast cancer cells13. These discrepancy implies that the EGFR-mediated multiple signal transduction pathways may have different effects on apoptosis.

How does EGFR regulate the growth and apoptosis in human hepatoma cells? EGF binds its receptor and stimulates a series of rapid response including phosphosylation of tyrosine residues within the EGFR itself and many other cellular proteins, hydrolysis of phosphatidyl inosital, release of Ca2+ from intracellular stores and morphological changes. EGFR plays a key role mediating the signal transduction. The deregulation of EGFR expression and function is supposed to disturb the signal pathways in the cells.

As a member of second messagers, calcium may be a sensitive target responsing to the modulation of EGFR activities. We have found that TG, an inhibitor of Ca2+ - ATPase on surface of endoplasmic reticulum induces a transient increase of intracellular Ca2+ in BEL-7404 cells and disturbs the response of intracellular Ca2+ following the stimulation with EGF, due to the depletion of the intracellular Ca2+ storage in endoplasmic reticulum14, 15. In this study, we compared the intracellular Ca2+ level and response to the induction of EGF and TG treatments in antisense EGFR transfected JX-1 cells and control vector transfected JX-0 cells. As shown in Fig 3, short-term treatment of 100 nM TG or 300 ng/ml EGF induced the release of Ca2+ in both JX-1 and JX-0 cells. The response was much weaker in JX-1 cells than in JX-0 cells by measuring fluorescence intensity. After EGF pre-treatment, JX-1 and JX-0 cells were still sensitive to TG induced stimulation followed by the release Ca2+ with a similar pattern as to TG treatment alone, but a much higher level of intracellular Ca2+ intensity was found in JX-0 cells than in JX-1 cells. However, TG pre-treatment obviously depleted the intracellular Ca2+ storage in the endoplasmic reticulum of the cells resulting in the loss of responsiveness to EGF induction in both JX-1 and JX-0 cells. These results suggested that the antisense EGFR vector transfected JX-1 cells might possess an decreased intracellular Ca2+ pool content in endoplasmic reticulum of the cells under the control of an unknown cascade reactions originating from the interrupted EGFR expression and function triggered by antisense EGFR sequence. It has been shown that the sustained elevation of cytosolic Ca2+ is capable of inducing apoptosis16. On the other hand, alteration of intracellular Ca2+ pool content has been considered to be linked to the control of cell growth17 as well as apoptosis18, 19. Our results in this report would support the notion that the alteration of intracellular Ca2+ pool content mediated by antisense EGFR sequence may interfere the signal pathways in regulating and enhancing the process of apoptosis in the JX-l cells. Therefore, the link between EGF/EGFR signaling and intracellular Ca2+ level in the induction of apoptosis of JX-1 cells as well as the exact role played by antisense EGFR remain an attractive target for the future studies.

Fig 3
figure 3

Effects of TG and EGF on intracellular Ca2+ level in JX-0 and JX-1 cells. Intracellular free Ca2+ levels were measured in fluo-3-1oaded cell populations as described in ref. 14. The peaks were recorded after the application of either EGF (300 ng/ml) or TG (100 nM) for 1 min. EGF-TG: after the pre-incubation with EGF (300 ng/ml) for 10 min, without washing, TG (100 nM) was added for 1 min before intracellular Ca2+ measurement. TG-EGF: after the pre-incubation with TG (100 nM) for 20 min, without washing, EGF (300 ng/ml) was added for 1 min before intracellular Ca2+ measurement.

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