Journal of Experimental & Clinical Cancer Research 2008, 27:87 http://www.jeccr.com/content/27/1/87 for 30 minutes before the addition of propidium iodide (PI), and then placed at 4°C for more than 15 minutes. Finally, the cell cycle distribution (G0/G1 phase, S, G2/M) and cell apoptosis rate was detected by FCM Measurement of the target genes by FQ-PCR K562 cells were collected from each group, washed with PBS three times. Total RNA was extracted from the cells according to the RNeasy Mini Protocol (Qiangen Biothch, Beijing). Each 1 μg total RNA from each group of K562 cells was reversely transcribed in a 20 μL reaction mixture which contained 0.5 μg oligo (dT)15 as primer by Super- Script II reverse transcriptase (Promage). The resultant cDNA was subjected to FQPCR. PCR was carried out in a total 25 μl reaction system, with the SYBR Green I PCR Reagent kit (Qiangen Biothech, Beijing) and a Lightcycler fluoresencent quantitative amplification analyzer (Bio- Rad, Hercules, CA). The reaction condition was 94°C for 5 min, 94°C 45s and 60°C 1 min, for 30 cycles. The fluo- rescence signal of the first 3 to 15 cycles was settled as flu- orescent background signal and the baseline was adjusted accordingly. Relative quantification of gene expression level was calculated by the following method: for each reaction to amplify the target gene and β-actin gene, the Ct value was determined. The ratio of of each target mRNA to β-actin mRNA was calculated by the formula 2-ΔCt. The specific primers' sequences for the target genes were shown in Table 1. Measurement of the target proteins by western blotting Western blot analysis was performed for detecting the expression level of specific protein in freshly washed K562 cells from each group. The protein concentrations of the cell lysate were measured by coomassie brilliant blue kit (Kang Chen Biotechnology, Beijing, China). Each 50 μg of the cell lysate was loaded onto a 10% SDS-PAGE gel, and run at 100 volts for 2 h. Cell proteins were transferred to nitrocellulose membrane, blocked with 5% milk in TBS (Tris-buffered saline with 0.1% Tween 20) for 1 h, then, Table 1: Primer sequence for FQPCR Primers BCR/ABL PTEN mTOR CyclinD1 P27kip1 β-actin Sequence F : 5'- GGAGCTGCAGATGCTGACCAAC-3' R : 5'- TCAGACCCTGAGGCTCAAAGT-3' F : 5'-ATACCAGGACCAGAGGAAACC-3' R : 5'-TTGTCATTATCCGCACGCTC-3' F : 5'- GAGCCAGTGTTCCCTCCAT-3' R : 5'- CAGTCAGCGGCCAGTCAT-3' F : 5'-CGATGCCAACCTCCTCAACGAC-3' R : 5'-CCAGCATCCAGGTGGCGACG-3 F : 5'-ACGTGAGAGTGTCTAACGG-3' R : 5'-AGTGCTTCTCCAAGTCCC-3' F : 5'- CTGGCACCACACCTTCTACAAT -3' R : 5'- AATGTCACGCACGATTTCCCGC -3' washed and incubated with the primary antibody for 1 h at room temperature or overnight at 4°C. The blots were washed and incubated with the horseradish peroxidase- conjugated secondary antibody, and developed with a chemiluminescent substrate, ECL Plus. Following devel- opment of the primary antibodies, the bounded immu- noglobulins were removed from the membranes by washing twice, each time for 15 minutes at room temper- ature in Restore Western blot Stripping Buffer. An autora- diograph was obtained, and protein levels were measured using a Fluors scanner and Quantity One software for analysis (Bio-Rad). Statistical analysis tatistical analysis was done by Student's t-test and Spear- man's test. Data was shown by mean ± SD of three inde- pendent experiments. The results from each group were compared with that of the corresponding control. P < 0.05 was considered as statistically significance. Results Anti-proliferation effect of PTEN transfection in K562 cells Our preliminary experiment and several published papers showed that K562 cells expressed PTEN mRNA and pro- tein at very low levels [8]. Therefore, K562 cells were trans- fected with Ad-PTEN-GFP or Ad-GFP in the present study. After the transfection, the difference in cell growth inhibi- tion rate between K562 cells from each group was not sta- tistically significant in the first 3 days. The maximal growth inhibition rate of 35.2% in Ad-PTEN-GFP trans- fected K562 cells was detected at the fifth day and that was statistically significant higher than that in Ad-GFP trans- fected K562 cells and that in control K562 cells (P < 0.01 Fig 1). PTEN transfection regulated the expression of specific genes and proteins in K562 cells After transfection of Ad-PTEN-GFP to K562 cells, the expression level of PTEN mRNA increased gradually, Product size Accession number from genebank 200 bp 101 bp 128 bp 143 bp 138 bp 382 bp AY789120 000314 NM_152756 NM_053056 NM_004064 NM_001101 Page 3 of 9 (page number not for citation purposes)