Transfection of promyelocytic leukemia in retrovirus vector inhibits growth of human bladder cancer cells.
OBJECTIVE
To construct a recombinant retrovirus vector carrying human promyelocytic leukemia (PML) cDNA and identify its expression and biology role in bladder cancer UM-UC-2 cells for future gene therapy.


METHODS
PML full-length cDNA was inserted into the EcoR I and BamH I site of pLXSN vector containing the long terminal repeat (LTR) promoter. The vector was identified by restriction enzyme digestion and then transfected into PA317 packaging cell line by calcium phosphate coprecipitation. PML cDNA was detected by polymerase chain reaction (PCR) and the protein was identified by laser confocal microscopy and Western blot in bladder cancer cells, respectively. The morphology was observed by inverted phase contrast microscope, and MTT assay determined growth curve of the bladder cancer cells.


RESULTS
Restriction enzyme digestion proved that a 2.1 kb PML cDNA was inserted into the pLXSN vector. PCR assay demonstrated that 304 bp fragments were found in UM-UC-2/pLPMLSN transfects. Laser confocal microscopy showed speck dots fluorescence in the UM-UC-2/pLPMLSN nucleus. A 90 kD specific brand was found by Western blot. MTT assay demonstrated the UM-UC-2/pLPMLSN bladder cancer growth inhibition.


CONCLUSIONS
The retrovirus pLPMLSN vector was successfully constructed and could generate high effective expression of human PML in bladder cancer cell UM-UC-2, suggesting that PML recombinant retrovirus have potential utility in the gene therapy for bladder cancer.
Li
Lei
L
Institute of Urology, First Hospital of Xi'an Jiaotong University, China.
He
Da-lin
DL
D045744:Cell Line, Tumor; D049109:Cell Proliferation; D018076:DNA, Complementary / Q000235:genetics; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D015473:Leukemia, Promyelocytic, Acute / Q000235:genetics*; D009363:Neoplasm Proteins / Q000235:genetics* / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D000071156:Promyelocytic Leukemia Protein; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D025521:Tumor Suppressor Proteins / Q000235:genetics* / Q000378:metabolism; D001749:Urinary Bladder Neoplasms / Q000378:metabolism / Q000473:pathology*
D018076:DNA, Complementary; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D000071156:Promyelocytic Leukemia Protein; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; C070576:PML protein, human