Different gene expression profiles of AD 293 and HEK 293 cell lines that show contrasting susceptibility to apoptosis induced by overexpression of Bim L

Bim is a pro-apoptotic member of the Bcl-2 protein family. Overexpression of Bim proved to be highly cytotoxic for diverse cells. The AD293 cell line is derived directly from the HEK293 cell line but has been transfected with a gene that can improve cell adherence. We found that there was almost no apoptosis seen in Bim L-transfected AD293 cells, but more than half of Bim Ltransfected HEK293 cells underwent apoptosis. Suppression subtractive hybridization was used to detect the different gene expression profile between these two cell lines. In 192 sequenced positive clones, there were 30 clones repeating twice or more. Ten genes were selected for identification by semi-quantitative RT-PCR. The transcripts of two adhesion-related genes (actin and parvin) and two apoptosis-related genes (cyclin 2 and protein phosphatase 1G) were upregulated in AD293 cells. These results suggest that the high expression of cell adhesion-related proteins might be responsible for the different apoptosis status after the transfection of Bim L. Our data provide candidate genes responsible for the different apoptosis sensitivity of these two cell lines. Further investigation on the differential expression profile between AD293 and HEK293 might improve our understanding of cell apoptosis mechanism.


InTRODuCTIOn
Apoptosis, the physiological process of cell death, is critical for modeling tissues and maintaining homeostasis in multicellular organisms (Kerr et al., 1972).Deregulation of apoptosis may cause diseases, such as cancers, immune diseases, and neurodegenerative disorders.Signaling for apoptosis occurs through multiple independent pathways that are initiated either from triggering events within the cell or from outside the cell.All apoptosis signaling pathways converge on a common mechanism of cell destruction that is activated by a family of cysteine proteases (caspases) that cleave proteins at aspartate residues (Strasser et al., 2000).Although the caspases represent a central point in apoptosis, their activa-tion is regulated by a variety of other factors.Among these, Bcl-2 family plays a pivotal role in caspases' activation, by this deciding whether a cell will live or die (Burlacu, 2003).The Bcl-2 protein family contains pro-and anti-apoptotic proteins that regulate the mitochondrial pathway of apoptosis (Antonsson, 2004;Vaux et al., 1988;Adams & Cory, 1998).The balance between these counteracting proteins presumably determines the cell fate (Oltvai et al., 1993;Thompson, 1995;White, 1996).
Bim is a 'BH3-only' pro-apoptotic protein of Bcl-2 family in the mitochondria pathway (O'Connor et al., 1998).To date, eleven isoforms of Bim have been reported (Mami et al., 2001;JinZhong et al., 2004).Three classical isoforms (Bim EL, Bim L and Bim S) contain a hydrophobic C-terminal region and a BH3 domain (O'Connor et al., 1998;Puthalakath et al., 1999).Resent research showed that Bim could induce apoptosis through direct interaction with Bax as well as through interaction with Bcl-2 and Bcl-X L (O'Connor et al., 1998;O'Reilly et al., 2000;Marani et al., 2002).To investigate the role of Bim in cell apoptosis, we transiently transfected HEK293 and AD293 cells with the plasmid pcDNA3.0-BimL. Overexpression of Bim L can induce apoptosis in about half of HEK293 cells but cannot induce AD293 cells' apoptosis.The AD293 cell line is derived directly from the HEK293 cell line by transfection with a gene that improved cell adherence (Graham et al., 1977).The different apoptosis susceptibility of these two cell lines to Bim L promotes us to pursue further study.
Here we report a differential gene expression profile obtained by a suppression subtractive hybridization between HEK293 and AD293 cells.

MATERIALS AnD METHODS
Cell culture, transfection, Western blot analysis and apoptosis assays.HEK293 and AD293 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 50 μg streptomycin/ml, and 50 IU penicillin/ml.The cultures were incubated at 37°C in a humidified atmosphere of 5% CO 2 .
HEK293 and AD293 cells were plated at a density of 2 × 10 5 cells per well.When the plates were about 75% confluent, cells were transfected with pcDNA3.0-BimL or pcDNA3.0using the Li-pofectamine agent (Invitrogene).Twenty-four hours after transfection, the cells were harvested.Apoptotic cells were quantified by flow cytometric analysis (APO-BRDU TM Apoptosis Assay Kit, Becton Dickinson) according to the manufacturer's recommendations.Total cell extracts were resolved by SDS/ PAGE.The expression of Bim L was detected by immunoblotting with a human Bim EL specific rabbit polyclonal antibody (Santa Cruz Biotechnology) followed by horseradish peroxidase-conjugated antirabbit antibody (Sigma-Aldrich).The proteins were visualized by using enhanced chemiluminescence immunoblotting detection reagents (Amersham).The blots were also developed with an anti-actin rabbit polyclonal antibody (Sigma-Aldrich) as a control.
Suppression subtractive hybridization between HEK293 and AD293 cells.AD293 and HEK293 cells were cultured in five 25 ml culture flasks.Cells were seeded at a density of 2 × 10 6 /ml.Total RNA was extracted by using Trizol agent (GIB-COL).mRNA was isolated by using Oligotex mRNA Kits (QIAGEN).The PCR cDNA subtraction kit and Advantage 2 DNA polymerase were purchased from Clontech.Both the mRNA of AD293 and HEK293 were used as driver and tester for constructing forward and reverse libraries.Muscle mRNA and Hae III-digested Φx 174 DNA testers and muscle mRNA driver were employed as the control panel.The SSH were performed with a standard procedure recommended by the manufacturer.Each of 192 independent clones of AD293 forward and reverse libraries was amplified with M13 consensus primers and immobilized on an Immobilon-Ny+ membrane (Millipore) with 50 ng/clone per spot.
The blots were detected with probes of the PCR products amplified with M13 consensus primers both from forward and reverse libraries.
The probes were labeled with (α-32 P)-dATP using a random primer DNA labeling kit (Worthington).The clone identity was verified by sequencing the corresponding plasmids with M13 consensus primers.All draft sequences were identified by Blast analysis against NCBI database (www.ncbi.nlm.nih.gov).

RT-PCR.
Transcripts of 10 different expressed genes between AD293 and

Gene identity
Primer sequence Expected amplified fragment length NM_014739 5'-gttgtgtgtgtgtgttaaaaggagc-3' 5'-atcaccctagcctactacactctg-3' AD293 and HEK293 cell lines susceptibility to apoptosis induced by overexpression of Bim L HEK293 cells were selected out to identify by semiquantitative RT-PCR.The first-strand cDNAs were synthesized using AMV Reverse Transcriptase (Promega).Then, 10 pairs of gene specific primers (Table 1) and Advantage 2 DNA polymerase (Clontech) were used to amplify the cDNAs, and G3PDH primers (Table 1) were used to amplify the same cDNAs as a control.The amplification conditions were as follows: 94°C 30 s, 65°C 30 s, 72°C 90 s, 30 cycles.The relative quantities of the specific PCR products were determined by densitometric analysis and normalized with the density of the G3PDH product.
The targeted PCR products were cloned into T-vector and sequenced with both M13 consensus primers.

Differential gene expression profile between AD293 and HEK293 cells
Using the PCR products from AD293 cells forward subtraction library as probes, we detected the forward and reverse subtraction library, respectively, and found that genes in these two libraries were significantly different (Fig. 2).The result indicated that the SSH we performed was credible.In 192 sequenced clones, there were 30 clones repeating twice or more.Among these clones, there were two cell adhesion-related genes, ten cell apoptosisrelated genes, two cell cycle-related genes and three neurodegenerative diseases-related genes (Table 2).
By performing semi-quantitative RT-PCR analysis, we found that among 10 up-regulated genes came from the AD293 forward library, the transcripts of 7 genes were clearly up-regulated.One of the ten had shared a similar expression level in both cell lines.In lane 3, there was a weak band in AD293 sample but no band in HEK293 sample.
All bands were verified by sequence analysis.An unexpected band of about 2 kb appeared in lane 8 of the AD293 sample.This turned out to be an intron containing fragment corresponding to a region of human cytoskeletal gamma actin gene, (ACTG1, M19283).The transcripts of the G3PDH control were almost of the same intensity in the two cell lines (Fig. 3).

DISCuSSIOn
HEK293 cells are human embryonic kidney cells transformed by sheared adenovirus type 5 DNA (Graham et al., 1977).The HEK293 cell line and its sublines are widely used in molecular and cellular research.AD293 cells constructed by the Stratagene Company for adenovirus transfection system are de- rived from the commonly used HEK293 cell line but by transfection with a gene that can improve cell adherence and plaque formation properties (Adeasy XL adenoviral vector system, catalog 240010).
Compared with HEK293 cells transfected with the empty pcDNA3.0plasmid, AD293 cells transfected with the same empty plasmid showed a lower apoptosis frequency.Moreover, AD293 cells also resist antibiotic G418 selection and serum deprivation (not show).Compared with transfected HEk293 cells, transfected AD293 cells expressed Bim L protein at an even higher lever (Fig. 1A).This prompted us to explore the differences between the AD293 and HEK293 cell line.
We performed a SSH to detect the gene expression difference between these two cell lines.Using PCR products from the AD293 forward subtraction library as probes, a dot blot was performed.In 192 positive sequenced clones, there were 30 clones repeating twice or more (Table 2).Among these clones, there were seven putative genes, six housekeeping genes, two cell cycle-related genes (cyclin L2 and FIN), three neurodegenerative diseases-related genes (kinesin family member 5C, transmembrane trafficking protein and DHCR24), two cell adhesion-related genes (actin and parvin), ten apoptosis related-genes (LOC203438, cytochrome c, caspase 6, BIRC2, STE20 homolog, BTF, PMSA3, ATPase, cathepsin D and G protein-coupled receptor kinase 6).
We performed an RT-PCR analysis of 10 genes involved to determine the reliability of the library produced by SSH.Both the dot blot and RT-PCR results suggested that the SSH library was reliable (Figs. 2, 3).All ten PCR products were verified by sequencing.As to the unexpected band of about 2 kb appearing in lane 8 of the AD293 sample, we speculated that this unexpected sequence in AD293 came from a plasmid containing ACTG1 (actin, gamma 1) which is tranfected into AD293 cells, an hnRNA or genomic DNA contamination (Erba et al., 1988).The RNA extraction of AD293 and RT-PCR of ACTG1 were preformed three times and the results showed no significant difference (not shown).
The loss of cell adherence results probably from the rearrangement of the cytoskeleton caused by proteolysis of actin or fodrin (Monteiro et al., 2004) and the loss of adherence ability in adherent cells is one reason for apoptosis initiation (Puthalakath et al., 2001).There were two cell adhesion-related genes (actin, parvin) up-regulated in AD293 cells.The high expression of these cell adhesion-related proteins might be responsible for the different apoptosis status after the transfection of Bim L. Our research was consistent with that of Collins and Fukazawa (Fukazawa et al., 2004;Collins et al., 2005).
There were five apoptosis-inhibiting genes up-regulated in AD293 cells.BTF encoded by NM_ 014739 can inhibit the transcription of bcl-2 apoptotic genes (Kasof et al., 1999).PSMA3 encoded by NM_002788 can prevent cell apoptosis through accelerating the degradation of apoptotic proteins (Wakako et al., 2001).NM_001695 and NM_001909 encode ATP6V1C1 and cathepsin D, respectively.The former is associated with the H + regulation of lysosome and is supposed to prevent apoptosis by inhibiting cytoplasm acidification (Izumi et al., 2003).The latter triggers Bax activation and Bax induces the selective release of mitochondrial AIF which is responsible for the early apoptotic phenotype (Bidere et al., 2003).cIAP encoded by NM_001166 may be a factor in the presumed anti-apoptotic system in human gastric cancer cells (Endo et al., 2005) and has been suggested to play important roles in the development of cervical and esophageal squamous cell carcinomas (Tanimoto et al., 2005).
AD293, HEK293 transfected with an adherence gene, presents higher expression of adherencerelated proteins and anti-apoptosis proteins.It resisted apoptosis signals such as over-expression of Bim L and growth factors deprivation.Our results suggest that the anti-apoptotic activity of enhanced cell adherence plays a role both in neutralizing proapoptotic molecules and in up-regulating expression of anti-apoptotic proteins.Further investigation might improve our understanding of cell apoptosis mechanism.

Figure 1 .
Figure 1.Different responses of AD293 and HEK293 to Bim L-mediated apoptosis.A. Western blot analysis of AD293 and HEK293 cells transfected with Bim L. The expression of Bim L was detected by immunoblotting with an anti-Bim polyclonal antibody.A parallel blot for actin served as loading control.B. Apoptosis assay of HEK293 and AD293 cells transfected with pcDNA3.0-BimL or pcDNA3.0.Each group repeated three times.

Figure 2 .
Figure 2. Dot blot of the SSH between AD293 and HEK293 cells.a, b) Using the PCR products from AD293 forward library as probes, AD293 forward clone blots were detected; c, d) using the PCR products from AD293 reverse library as probes, HEK293 forward clone blots were detected; e, f) AD293 forward clone blots were detected by probes from AD293 reverse library; g, h) HEK293 forward clone blots were detected by probes form AD293 forward library.