Reagents

Dosper liposome transfection kit and TUNEL kit were purchased from the Roche Company. Western blot kit, polyclonal anti-HA antibody was bought from Santa Cruz. MTT was purchased from Huamei Company. G418 was Clontech Company's product. Plasmid maxiiprep kit was purchased from Omega. Annexin V-FITC and PI were obtained from Becton Dickinson. CDDP and ADM were bought from the Qilu Company and Hisun Company respectively. The drugs were dissolved according to the manufacturer's instructions and diluted in culture medium before addition to the cell cultures.

Plasmid

The plasmids pcDNA3-HA-p53 and pcDNA3-HA-p73αwere a kind gift from Prof. Gerry Melino and Dr. Vincenzo De laurenzi in University of Rome‘Tor Vergata'(Rome, Italy).The plasmids were used to generate cell lines that inducible express p53 or p73α. Both P53 and P73α protein were tagged at their N-termini with an influenza hemagglutinin (HA) peptide. Empty vector pcDNA3 was provided by Professor Dirk Gruendemann in University of Cologne(Cologne, Germany). Plasmids were amplified in coli. DH5α, and the extraction, purification and identification of the plasmid followed the instruction of the kit and the routine.

Cell culture, transfection and screening

The human NSCLC cell lines A549 was donated by Doctor Shi Tong-dong in the Immunological Academe of the Third Military Medical University (Chongqing, China). The A549 cells have wild-type p53 but do not express endogenous p73 protein. Cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, and 100 units/ml penicillin and 100 µg/ml streptomycin. Cells were culture in a 5% CO₂ incubator at 37°C, and the medium was changed every 3 days. Cells were split as needed with 0.25% trypsin-0.02% EDTA. All of the transfections were performed in six-well plates with Dosper according to the manufacturer's instructions (DNA/Dosper ratio 1:3). The day before transfecting, Approx 1x10⁵ A549 cells were seeded on every well in 2 ml medium, at 70-80% confluence, we used Dosper reagent to perform transfeciton. 2 µg plasmid DNA and 6 µl Dosper reagent were incubated with cells for 6 hours. The cells were divided into 4 groups, including the non-transfection group (A549), the group transfected with pcDNA3 (A549/pcDNA3), the group transfected with pcDNA3-HA-p53 (A549/p53) and the group transfected with pcDNA3-HA-p73α(A549/p73α). After the growth of the transfected cells for 48 hours, we splited the cells in every well at the cell density of 1/3, and screened out the resistant clones with 1 000 µg/ml G418, non-transfected cells as the control. The screening was sustained by the G418 medium 400 µg/ml and the subculture of the cells was continued and amplified. Individual clones were screened for inducible expression of P53 or P73αprotein by western blot analysis.

Western blot analysis for P53 or P73α expression

Western blot analysis was performed using the ECL western blot detection system and protocol. Briefly, cells were scraped from the plates, wash with PBS, and were lysed with RIPA solution [1% NP-40, 0.5% sodium deoxycholate, 10% sodium dodecylsulfate (SDS), 3 µl/ml aprotinin and 5 µg/ml leupeptin in PBS, pH7.4]. The total protein was obtained by centrifuging at 10 000 rpm (10 600 g) at 4 °C; 10 µg/slot of proteins were boiled in a sample buffer for 1 min at 100 °C, electrophoreseed in 10% SDS/polyacrylamide gel and than transferred to PVDF membranes. Nonspecific antibody bindings were blocked by preincubation of the membranes with 5% non-fat milk overnight. Then the membranes were incubated for 1 h with the polyclonal rabbit anti-human HA antibody (1:200), followed by further incubation 1 h with goat anti-rabbit IgG (1:2500). After each antibody incubation the membranes were washed three times by Tris-buffer saline. To visualize the protein bands, enhanced chemiluminescence western blot detection reagents were used and the membranes were exposed to X-ray film.

MTT assay

MTT assay is also used for the measurement of cytotoxicity of drug alone or in combination treatments. Exponentially growing cells of A549, A549/pcDNA3, A549/p53 and A549/p73αwere harvested and resuspended to a final concentration of (1~2)x10⁵ cells/ml in fresh medium. Cell suspensions (200 µl) were dispensed into wells of 96-well culture plates. After 24 h culture at 37 °C for cell adherence to the plate, the solutions of CDDP or ADM were simultaneously added to the medium, the finally concentrations were 0, 0.01, 0.05, 0.25, 1.25, 6.25 and 31.25 µmol/l, respectively, and subsequently incubated 24 hours. Then 20 µl MTT (5 mg/ml) was added to each well then incubated for 4 hours under the same condition. After incubation, culture medium in each well was discarded and replaced with 200 µl DMSO. After 10 minutes' shaking, the absorbance of each well was determined by a spectrophotometer with a 490nm wavelength. The wells treated by the complete medium without chemotherapeutic drug served as a control. The percentage of cell viability was calculated by multiplying the ratio absorbance of the sample versus the control by 100. Chemotherapeutic drugs' IC₅₀ was determined as a chemotherapeutic drugs' concentration showing 50% cell growth inhibition as compared with control cell growth. The experiments were repeated in triplicate.

Apoptosis assay

Cell apoptosis was monitored by three methods. First, cell apoptosis was measured by flow cytometry. exponentially growing cells of A549, A549/pcDNA3, A549/p53 and A549/p73α were divided into treatment group and non-treatment group, respectively. Each bottle was then incubated for 48 h with cDDP (final concentration 6.25 µmol/L) or ADM (final concentration 0.25µmol/L). Then both floating dead cells in the medium and live cells on the wall of bottle were collected and washed twice with cold PBS. The cells were resuspended in 0.1 ml of PBS to a density of 1x106/ml. Cells were stained with 5 µl Annexin V-FITC and 10 µl PI (50 mg/L), and incubated at room temperature for 30 min in the dark. Then 400 µl staining buffer was added to bring the total volume to 500 µl prior to FCM analysis. At least 10 000 cells of each sample were analyzed each time. Second, cell apoptosis can also be reflected by TUNEL analysis. In brief, after the same treatment schedule described above. Slides with cells were then washed, and fixed for 30 min in 4% paraformaldehyde, incubated in 3% H₂O₂ in methanol for 10 min at room temperature and then permeabilized with 0.1% Triton X-100 for 2 min. After incubation with reaction buffer containing terminal deoxyribonucleotidyl transferase according to the manufacturer's protocol (Roche Molecular Biochemicals), the slides were stained with ABC reagent. After this, the slides were incubated with DAB/H₂O₂ solutions and retained with hematoxylin after complete washing. Data shown are representative of three independent experiments. Third, the harvested cells were lysed in a solution containing 100 mmol/L NaCl, 10 mmol/L Tris, 25 mM ethylenediaminetetraacetic acid (EDTA), and 0.5% sodium dodecyl sulfate. After the centrifugation, the supernatant was incubated with 300 µg/ml proteinase K for 3 hours at 65°C and extracted with phenol-chloroform. The aqueous layer was treated with 0.1 volume of 3 mol/L sodium acetate, and the DNA was precipitated with 2.5 volumes of ethanol. After treatment with 100µg/ml RNase A for 1 h at 37°C, the sample was electrophoresed on a 2% agarose gel and visualized by ethidium bromide staining and ultraviolet transillumination.

Clone formation assay

Cells were plated in log phase into six-well plates with 5 ml medium and grown at 37 °C and with 5% CO₂ silently. After 48 h culture for cell adherence to the plate, rinsed with fresh medium, and then CDDP (final concentration 6.25 µmol/L) or ADM (final concentration 0.25µmol/L) were added to each well. After cells were incubated for 48 h with chemotherapeutic drug, Cells were then washed twice with prewarmed PBS, trypsinized, diluted and 500 cells per treatment were seeded into six-well plates in triplicate with chemotherapeutic drug-free medium. The medium was changed every 5 days. Colonies were allowed to grow for 10±14 days. The medium was discarded and each well was washed twice by PBS carefully. The cells were fixed about 10 minutes by 1:3 acetic-methanol, and then stained with Giemsa for 10±30 minutes. Finally, positive colony formation (more than 50 cells/colony) was counted. The survival fraction for each cell line was expressed as the ratio of plating efficiency of treated cells to that of untreated control cells.

Statistical analysis

The data were analyzed with SPSS 10.0. The results were expressed as the mean±standard deviation x(_)±s. Statistical significance was determined by t test. A P value of 0.05 or less was considered statistically significant.

Exogenous protein stable expression achieved in A549 cells

By tagging p73αand p53 with an HA epitope, the relative amount of the exogenous P73α and P53 proteins can be qualitative and quantified by Western blot analysis with rabbit anti-HA polyclonal antibody. The expression of HA was detected in cells transfected with pcDNA3-HA-p53 and pcDNA3-HA-p73α by western blot (as shown in Fig 1), demonstrating that the transfection of pcDNA3-HA-p53 and pcDNA3-HA-p73α were successful. The normal level of p73 is undetectable in A549 cells. However, a high level of expression of P73α protein was achieved in A549/p73αby western blot assay. Plasmid pcDNA3 was not tagged by HA epitope, but it could express neo gene. Therefore, we used G418 to screen A549 and A549/pcDNA3 cells. Screened by G418 medium 1 00 mg/L for 4 days, most of the A549 cells died, while A549/pcDNA3 cells grew normally, which proved that the transfection of pcDNA3 was successful.

Figure 1: Detection of exogenous P73-HA protein in parent and transferring cell line

MTT assay

Similar viability of A549 and A549/pcDNA3 acted by the same drug concentration were observed, there was only slight growth inhibition in the cells transfected with pcDNA3-HA-p53. However, the viability of A549/p73α was significant lower than those of A549, A549/pcDNA3 and A549/p53. This suggested that p73α was more cytotoxic than pcDNA3 and p53 in A549 cells, consistent with a p73α specific effect. 6.25µmol/L CDDP or 0.25µmol/L ADM could only damage A549 or A549/pcDNA3 cells under 50%, a subtoxic concentration. By contrast, A549/p73α cells responded to the subtoxic concentration chemotherapetutic drugs (6.25 µmol/L CDDP or 0.25 µmol/L ADM) sensitively, over 50% cells were killed (as shown in Fig 2). Furthermore, the concentrations of agents that caused 50% inhibination of growth (IC₅₀) was determined, p73α could cause a decrease in IC₅₀ of cDDP and ADM (as shown in Tab.1). The cell growth inhibitory effects of the combined treatment were synergistic and more significant compared with p73α or chemotherapeutic drug alone.

Figure 2: Alteration of cisplatin and adriamycin chemosensitivity caused by the transfer of p73α or p53 gene in A549 cell line

Table 1: IC₅₀ Vaules of Cisplatin and Adriamycin in A549 Cells before and after p73 Gene Transfection

Apoptosis assay

Flow cytometry showed that much more apoptotic cells were observed in the group treated by the combination compared with those treated by p73α or chemotherapeutic drug alone (as shown in Fig 3).

Figure 3: Apoptosis was measured by flowcytometry analysis of Annexin V-FITC double-labeled cells

A significant difference (P<0.01) was observed between p73α gene transfect or chemotherapy alone and a combination of the two approaches (as shown in Tab.2).

Table 2: Apoptosis Index by Flowcytometry Analysis of A549 Treated with Cisplatin or Adriamycin before and after Gene Transfection( ±s)

Compared to A549 cells, the apoptosis rate increased 2.4 and 2.9 fold after treated by 6.25µmol/L CDDP or 0.25µmol/L ADM respectively. However, the apoptosis rate of the A549/p73α increased 8.2 and 9.1 fold after treated by 6.25µmol/L CDDP or 0.25µmol/L ADM respectively. Thus, there was significant apoptosis following p73α gene transfer, subsequent chemotherapy made the process even more effective. To examine this further, we performed TUNEL assay. The results are consistent with the data obtained by the Flow cytometry assay (as shown in Fig 4).

Figure 4: The CDDP(6.25 µmol/L) induced apoptosis of A549 cells before and after p73 gene transfection was measured by TUNEL.The Apoptosis index was significantly higher after p73 gene transfection.A: Apoptotic cells are brown and dispersed(x400).B. before p73 gene transfection(x200). C. after p73 gene transfection(x200).

To confirm the finding that p73α gene could enhance the apoptotic response induced by chemotherapeutic drugs, DNAs were isolated from A549, A549/p73α or A549/p53 cells treated with or without chemotherapeutic drugs and electrophoresed for DNA ladder formation. Using the same treatment schedule described above, the characteristic apoptotic DNA ladder was observed only after combined treatment with p73α and chemotherapy, but not with either agent alone (as shown in Fig 5).

Figure 5: DNA fragmentation in parent and transferring cell line 2% agarose gel electrophoresis of DNA

Clone formation assay

We examined the effect of p73α expression on cell biological behavior change by colony formation assay. After incubated for 48 h with 6.25 µmol/L CDDP or 0.25 µmol/L ADM, transfection of p73α result in significant or dramatic decrease in the number of colonies compared with A549, A549/pcDNA3 or A549/p53 (as shown in Fig.6).

Figure 6: The colony formation ratio of H1299 treated with CDDP or ADM before and after gene transfection

Compared to their respective non-treatment cells, the clones of A549, A549/pcDNA3, A549/p53 and A549/p73αcells exposured to 6.25 µmol/L CDDP or 0.25 µmol/L ADM decreased 37.4%, 39.6%, 46.7%, 76.3% and 33.5%, 33.8%, 44.4%, 80.5%, respectively. p73αgene transfection significantly inhibited the cells' clonogenicity after the treatment with chemotherapeutic drugs. A statistically significant difference (P<0.01) was observed between p73α transfer or drug exposure alone and a combination of the two treatments. However, A549/p53 partially retained their capacity.

Acknowledgments

We thank Prof. Gerry Melino and Dr. Vincenzo De laurenzi for providing the plasmids pcDNA3-HA-p53 and pcDNA3-HA-p73α.