[Adenoviral-mediated expression of PTEN cDNA induces apoptosis in endometrial carcinoma cells].
OBJECTIVE
To investigate whether the human endometrial carcinoma cells, RL95-2 infected with recombinant Ad-PTEN can steadily produce PTEN protein and enter apoptosis.


METHODS
The recombinant adenovirus containing PTEN cDNA was constructed using the method of homologous recombination in bacteria. The viral titer was examined by plaque assay and the expression of PTEN protein was detected by western blot assay. The apoptosis of RL95-2 cells was evaluated as following: flipping of membrane phosphatidylserine (PS) and identification of activating caspase-3 positive cells was determined by flow cytometer (FCM), and furthermore genomic DNA fragmentation was detected by agarose electrophoresis.


RESULTS
The recombinant adenovirus encoding PTEN cDNA was successfully constructed, and viral titers of Ad-PTEN were 5 x 10(9) pfu/ml. After infected by Ad-PTEN, the expression of PTEN protein was steady in human RL95-2 cells. After infected by Ad-PTEN for 24, 48, 72 and 96 h, the relative cell number of membrane PS flipping were (6.09 +/- 1.01)%, (9.98 +/- 2.17)%, (11.74 +/- 2.65)%, (27.69 +/- 8.67)%, which significantly increased than control group (P < 0.05), the relative cell number of activated caspase-3 positive were (2.6 +/- 0.5)%, (18.0 +/- 4.4)%, (21.8 +/- 5.1)%, (33.7 +/- 9.9)%, respectively, which significantly increased than control group (P < 0.05), and genomic DNA fragmentation was verified also.


CONCLUSIONS
The recombinant Ad-PTEN vector is constructed successfully and the expression of specific PTEN is steady in RL95-2 cell line. The expression of PTEN induces RL95-2 cells to apoptosis. PTEN gene may be a novel therapeutic target in endometrial carcinoma.
Yang
Pei-li
PL
Department of Obstetrics and Gynecology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China.
Liu
Yu-huan
YH
Cui
Ying
Y
Cai
Zai-long
ZL
Mao
Ji-fang
JF
D000256:Adenoviridae / Q000235:genetics*; D017209:Apoptosis / Q000235:genetics* / Q000502:physiology; D015153:Blotting, Western; D053148:Caspase 3 / Q000378:metabolism; D045744:Cell Line, Tumor; D053938:DNA Fragmentation; D018076:DNA, Complementary / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D016889:Endometrial Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D005260:Female; D005434:Flow Cytometry; D005822:Genetic Vectors; D006801:Humans; D051059:PTEN Phosphohydrolase / Q000235:genetics* / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
D018076:DNA, Complementary; D051059:PTEN Phosphohydrolase; D053148:Caspase 3