Adenovirus-mediated expression of both antisense ODC and AdoMetDC inhibited colorectal cancer cell growth in vitro.
OBJECTIVE
To construct a recombinant adenovirus that can simultaneously express both antisense ornithine decarboxylase (ODC) and adenosylmethionine decarboxylase ( AdoMetDC ) and detect its inhibitory effect on the intracellular polyamine pool and colorectal cancer cell growth.


METHODS
A 205-bp cDNA of AdoMetDC was reverse-inserted into recombinant pAdTrack-ODCas vectors and recombined with pAdEasy-1 vectors in AdEasy-1 cells. Positive clones were selected and transfected into the packaging cell HEK293 after they were linearized by PacI. Green fluorescent protein expression was used to monitor the process of adenovirus packaging. The ODC and AdoMetDC protein levels were identified by western blotting, and intracellular polyamine content was detected by reverse-phase high performance liquid chromatography. A viable cell count was used to determine the growth of HT-29 cells with or without exogenous polyamine.


RESULTS
Sequencing confirmed that AdoMetDC cDNA was successfully ligated into the pAdTrack-ODCas vector. GFP expression in 293 cells during virus packing and amplification was observed by fluorescence microscopy. Western blotting demonstrated that both ODC and AdoMetDC were downregulated by Ad-ODC-AdoMetDCas, and consequently 3 kinds of polyamine (putrescine, spermidine and spermine) were reduced to very low levels. HT-29 cell growth was significantly inhibited as compared with control conditions, and growth arrest was not reversed by exogenous putrescine.


CONCLUSIONS
The successfully constructed recombinant adenovirus, Ad-ODC-AdoMetDCas, blocked polyamine synthesis and has therapeutic potential for treating colorectal cancer in vitro.
Zhang
Bing
B
Institute of Biochemistry and Molecular Biology, School of Medicine, Shandong University, Jinan 250012, China.
Liu
Xian-xi
XX
Zhang
Yan
Y
Jiang
Chun-ying
CY
Teng
Qing-shan
QS
Hu
Hai-yan
HY
Wang
Wei
W
Gong
Lei
L
D012437:Adenosylmethionine Decarboxylase / Q000235:genetics / Q000378:metabolism*; D000256:Adenoviridae / Q000235:genetics; D002460:Cell Line; D049109:Cell Proliferation; D004622:Embryo, Mammalian; D005822:Genetic Vectors; D019073:HT29 Cells / Q000378:metabolism / Q000473:pathology*; D006801:Humans; D007668:Kidney / Q000166:cytology; D020319:Oligodeoxyribonucleotides, Antisense; D009955:Ornithine Decarboxylase / Q000235:genetics / Q000378:metabolism*; D011073:Polyamines / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D020319:Oligodeoxyribonucleotides, Antisense; D011073:Polyamines; D011994:Recombinant Proteins; D009955:Ornithine Decarboxylase; D012437:Adenosylmethionine Decarboxylase