Journal of Experimental & Clinical Cancer Research 2008, 27:87 http://www.jeccr.com/content/27/1/87 transcriptional modifications and GpG island methyla- tion [3-8]. mTOR (mammalian target of rapamycin) gene locates in the downstream of PI3K/Akt signal pathway, encoding a serine/threonine protein kinase which is considered as an oncoprotein. Studies showed that PI3K/Akt/mTOR and MAPK and FAK signaling pathways and their downstream partners were activated in leukemia, inducing the decon- trolled proliferation and enhancing the invasiveness of leukemia cells [9,10]. PTEN inhibits the activation of the above mentioned pathways via its lipid phosphatase and protein phos- phatase activities [8]. Rapamycin (RAPA), a triene mac- rolide antibiotic, has anti-mTOR activity by binding mTOR to interfere the interaction of mTOR with other tar- get proteins in the signaling pathway. Targeting mTOR signaling pathway can be a new strategy for the treatment of leukemia [10-13]. Several studies showed that down-regulation of PTEN mRNA expression and protein synthesis, and PI3K/Akt/ mTOR pathways were activated in the bcr/abl fusion pro- tein positive K562 cells. However, the relationship between the high expression of PTEN and the inactivation of mTOR signal pathway and their effects on the prolifer- ation of K562 cells were not clear. The present study was designed to transfect K562 cells with recombined adeno- virus-PTEN vector containing green fluorescent protein (Ad-PTEN-GFP) or Ad-GFP vector followed by the treat- ment of the cells with or without rapamycin. The regula- tory mechanism of PTEN and rapamycin on mTOR signaling pathway in K562 cells was then explored and the growth inhibition effect of this treatment on K562 cell was analyzed. Materials and methods Main reagents Chemicals were purchased from Sigma-Aldrich and Qian- gen Biotechnology (Qiangen, Beijing). Antibodies to PTEN, Akt, p-AKT (serine 473), GAPDH were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Second- ary antibodies for western blot analysis (horseradish per- oxidase-conjugated anti-mouse Ig) were purchased from Beijing Ding Guo biotechnology CO. LTD. Rapamycin (RAPA) was from North China Pharmaceutical Group Corporation. Other reagents were of analytical grade. Cell culture Human embryonic kidney cell line 293A cells were cul- tured in the medium of HD DMEM, supplemented with 10% fetal bovine serum and used for adenovirus amplifi- cation. K562 cells, a cell line established from a chronic myelogenous leukemia patient in blast crisis, were cul- tured in RPMI1640 supplemented with 10% fetal bovine serum, glutamine (2%), penicillin (100 U/ml) and strep- tomycin (100 μg/ml) at 37°C in a humidified atmosphere with 5% CO2. Exponentially growing K562 cells were seeded into flat-bottomed tissue-culture plates at the den- sity of 5 × 105 cells/ml and cultured overnight Cell transfection with Adenovirus Vectors The first generation of recombinant adenovirus was gen- erated by Jikai biological CO.LTD, Shanghai. The particle titers of the adenoviral stocks were typically 1 × 10E9 plaque-forming units per milliliter (pfu/mL). Adenovirus amplification was propagated in 293A cells for several times to obtain high-titer stocks, as determined by the plaque assay. Viral stocks of 1 × 10E9 pfu/mL were kept at -80°C. Adenovirus vectors expressing the transgene PTEN, containing green fluorescent protein (Ad-PTEN-GFP) or empty vectors (Ad-Vector) were used to transfect the K562 cells. At the condition when multiplicity of infection (MOI) was 200, K562 cells were co-cultured with Ad- PTEN-GFP or Ad-GFP at 37°C, in a humidified atmos- phere with 5% CO2, for 2 h. The transfection efficiency was detected directly by FMC for the expression ratio of green fluorescent protein. Measurement of the effects of PTEN gene-transfection on K562 cells From the first day to the seventh day after the transfection, cell growth inhibition rate of the transfected K562 cells from each group was measured by MTT method and FCM, the mRNA expression levels of PTEN, mTOR, cyclinD1, P27kip1 and BCR/ABL fusion gene in the transfected K562 cells were detected by FQ-PCR and the protein levels of PTEN, Akt, p-Akt were measured by western blotting. Measurement of growth inhibition effect of rapamycin on K562 cells Raparamcin (RAPA) was dissolved in DMSO and then added to the culture medium of the transfected K562 cells or control K562 cells at the final concentrations of 0, 1, 5, 10, 20, 50 and 100 nmol/L. The effects of rapamycin, together with PTEN transfection, on cell proliferation, apoptosis and cell cycle were detected by MTT assay and FCM when the K562 cells were treated with rapamycin for 12, 24, 48 or 72 hours. The mRNA expression levels of PTEN, mTOR, cyclinD1, P27kip1 and BCR/ABL fusion gene in the rapamycin reated and PTEN transfected K562 cells were detected by FQ-PCR and the protein levels of PTEN, Akt, p-Akt by western blotting. Cell cycle distribution assay For the cell cycle analysis, K562 cells (at least 1 × 105 cells) from each group were collected at each culture-ending point, washed and fixed with 70% ethanol, placed at 4°C overnight. Then, the cells were treated with RNA enzyme Page 2 of 9 (page number not for citation purposes)