The human gastric cancer cell lines, MKN-45 (wild-type p53) and MKN-28 (mutant type p53), were obtained from the Typical Culture Center of Wuhan University (China). Cells were maintained at 37?C in a humidified atmosphere of 5% CO₂ in RPMI 1640 (Gibco PRL) supplemented with penicillin/streptomycin (100 units/ml and 100 ?g/ml, respectively) and 10% fetal calf serum (FCS). Restriction enzymes and DNA recovery kits were purchased from Promega Company. T₄ DNA Ligase was purchased from the TaKaRa company. Propidium iodide was purchased from the Sigma company. TUNEL and caspase-3 activity detection kits were purchased from the Boster and Clontech companies respectively.

Plasmid pET-BAK was kindly provided by Dr. Patty Wendel (Cold Spring Harbour, USA), which contained the BAK cDNA (633 bp) between the restriction sites for XhoI and HindIII. Prof. Shen Qu (Department of Biochemistry, Tongji Medical College, China) provided the eukaryotic vector pcDNA₃. XhoI and Hind III digested the plasmids, pET-BAK and pcDNA₃, to recover the BAK gene (633 bp) and linear vector fragments (5.4 Kb) respectively. The ligation reaction was conducted with T₄ Ligase. According to the physical maps of both the BAK gene and vector, XhoI, HindIII, XhoI and HindIII digested the recombinant respectively, for restrictive enzyme reaction assay.

For both MKN-45 and MKN-28 cell lines, untransfer control, pcDNA₃ and pcDNA-BAK transfer groups were designed. Gene transfer was conducted with Lipofectamine 2000 (Gibco company, USA), according to the suggestion of the manufacturer. Cellular BAK mRNA expression levels were assayed by reverse transcription polymerase chain reaction (RT-PCR). Cellular total RNA extraction was conducted with TRIzol (Life Technologies company). After the reverse transcription reaction, PCR amplification reaction was conducted with the following primers: BAK (502 bp) upstream 5'-CTGCCCT CTGCTTCTGA-3', downstream 5'-CGTT CAGGATGGGACCA-3'. ?-Tubulin (295 bp) upstream 5'-CCCGTCTTCAGGGTCTCTTG-3', downstream 5'-TTAAGGTAAGTGTAGGTTGGG-3'. ?-Tubulin served as an internal control for PCR. Amplification products were separated with 1% agarose electrophoresis and observed under ultraviolet light. The brightness ratios between BAK and ?-Tubulin were evaluated with gel computer image system (Gel Doc 1000, Bio-Rad company). Cellular BAK protein expression levels were assayed via Western Blotting. The extraction, quantification and separation of proteins were conducted according to Molecular Cloning. Blots were incubated sequentially with 1% nonfat dry milk, mouse monoclonal anti-BAK antibody (Boster Company) and goat radish peroxidase-conjugated antimouse immunoglobulin G, and evaluated using an ECL Western blotting kit. ?-Tubulin served as an internal control for the immunoblot. Protein band intensities were determined densitometrically using the Computer Imaging System.

The MKN-45 and MKN-28 cells were seeded at 3 ? 10⁴/ml density in 96-well chamber slides (each group had five wells). After transferring for 1d, 3d and 5d, cellular growth was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-dimethyltetrazolium bromide (MTT, Clontech) colorimetric method. Each well was given 20 ?l of 0.5% MTT and cultured for another 4 h. The supernatant was discarded, then the pellet was given 100 ?l of dimethyl sulfoxide. When the crystal dissolved, the optical density A values of the slides were read on an enzyme-labeled Minireader II at 570 nm. Cellular proliferation inhibition rates are calculated as (%) = (1-average A_570nm value of experimental group/average A_570nm value of untransfer control group) ? 100%.

After transferring for 3d, 2 ? 10⁶ cells from the above groups were collected. Cells were washed twice with 0.01 mol/L phosphate-buffer saline (PBS) and fixed in 70% ethanol overnight at 4?C. Then the cells were washed once with PBS, digested by 200 ?l RNase (1 mg/ml) at 37?C for 30 minutes, and stained with 800 ?l propidium iodide (50 ?g/ml) at room temperature for 30 minutes. The DNA histograms were assayed by flow cytometry (Becton Dickson company) and analyzed with CELLQEST software.

Cancer cells from the groups above were collected, rinsed by PBS, fixed using 2.5% glutaraldehyde for 30 minutes, and then washed with PBS. After routine embedment and slicing, cellular ultrastructure was observed under an electronic microscope. The TdT-mediated dUTP-biotin nick end labeling (TUNEL) method detected the apoptotic rates. After blocking with 0.3% H₂O₂ for 30 min, slices were digested by Proteinase K (20 mg/L) for 20 min, then incubated with the TUNEL reaction solution at 37?C for 60 min. Then, the slices were incubated with peroxidase-conjugated antibody at 37?C for 30 min, stained with diaminobenzidine (DAB) and counterstained using haematoxylin. Instead of the TUNEL reaction solution, PBS was used as a negative control. Under a light microscope, the volume of the apoptotic cells became smaller, with shrinking nuclei and a specific palm-yellow color in the chromosome.

Cellular caspase-3 protein expression determinations came from Western Blotting (as the same method above). 2 ? 10⁵ cells from above groups were collected, 50 ?l of cellular lysis buffer was added, and then they were incubated on ice for 10 min. After centrifugation (12000 rpm) at 4?C for 3 min, the supernatant was collected and added sequentially with 50 ?l 2 ? Reaction Buffer, 5 ?l 1.0 mmol/L caspase-3 substrate DEVD-pNA, and incubated at 37?C for 1 h. After being transferred into 96 wells, the optical density A values of the slides were read on enzyme-labeled Minireader II at the wave length of 405 nm (A_{405 nm}), which is the relative activities of caspase-3.

Data analysis was performed using SPSS 10.0 statistical software.

The eukaryotic expression vector, pcDNA-BAK (6.0 Kb), was constructed. Electrophoresis, on a 1% agarose gel, showed the BAK cDNA insertion reactions into the recombinant were successful (Fig. 1A). After electrophoresis of RT-PCR products, BAK amplification bands could be observed in both untransfer and pcDNA₃ transfer groups. The difference between them is not significant (P>0.05). After transferring with pcDNA-BAK for 3d, BAK mRNA levels in MKN-45 and MKN-28 cells were significantly increased (P<0.01) (Fig. 1B). Western Blotting and densitometric analysis indicated BAK protein bands brightness of pcDNA-BAK transferred MKN-45 and MKN-28 cells were significantly higher than those of untransferred and pcDNA₃ transferred cells respectively (P<0.01) (Fig. 1C).

In untransfer control group, the A_570nm values were increased with the incubation time, indicating the active in vitro growth of MKN-45 and MKN-28 cells. After transferring with pcDNA-BAK for 1d~5d, the growth activities of MKN-45 and MKN-28 cells were both reduced to variable extents, with their inhibitory rates being 10.25%~32.41% (P<0.01), 11.54%~33.45% (P<0.01), respectively (Fig. 2).

On histograms of flow cytometry (Fig. 3), the S phase ratio in pcDNA-BAK transferred cancer cells was significantly lower than that of untransferred control, while cell cycles were arrested at the G₀/G₁ phase. In both pcDNA-BAK transferred MKN-45 and MKN-28 cells, sub-diploid DNA peak, corresponding to the apoptotic cell fraction, could be detected.

After transferring with pcDNA-BAK, parietal MKN-45 and MKN-28 presented characteristic morphological changes of apoptosis, such as nuclear shrinking, chromatin congregating around the nuclear membrane, reduction of cell volume and integrity of nuclear membrane, which could be viewed under an electronic microscope (Fig. 4A). The TUNEL assay indicated that the apoptosis rates of untransferred MKN-45 and MKN-28 cells were 4.7%, 4.2% respectively after being cultured for 3d. After pcDNA-BAK transfer for 3d, the apoptosis rates of MKN-45 and MKN-28 cells were 21.4%, 20.1% respectively (P<0.01). (Fig. 4B).

After pcDNA-BAK transfer, the 20 k Da (p20) and 17 k Da (p17) protein bands, subunits of caspase-3, could be detected in MKN-45 and MKN-28 cells (Fig. 5A). The A_{405 nm} values in untransferred MKN-45 and MKN-28 cells were 0.055 ? 0.008, 0.045 ? 0.010 respectively. After transferring with pcDNA-BAK for 3d, their cellular A_{405 nm} value were increased to 0.245 ? 0.012 (P<0.01), 0.238 ? 0.011 (P<0.01) (Fig. 5B).

We thank Dr. Patty Wendel (Cold Spring Harbour Laboratory) for her kind gift of the BAK gene. We also appreciate Prof. Dechun Li (Johns Hopkins University School of Medicine) for his great help in the preparation of this manuscript.