S-Adenosyl-L-methionine decarboxylase (SAMDC) is a key enzyme in the polyamines biosynthesis, thus is essential for basic physiological and biochemical processes in plant. In the present study, a full length cDNA of DoSAMDC1 gene was obtained from symbiotic germinated seeds of an endangered medicinal orchid species Dendrobium officinale, using the rapid amplification of cDNA ends (RACE)-PCR technique for the first time. The full length cDNA was 1 979 bp, with three open reading frames, i.e. tiny-uORF, small-uORF and main ORF (mORF). The mORF was deduced to encode a 368 amino acid (aa) protein with a molecular mass of 40.7 kD and a theoretical isoelectric point of 5.2. The deduced DoSAMDC1 protein, without signal peptide, had two highly conserved function domains (proenzyme cleavage site and PEST domain) and a 22-aa transmembrane domain (89-110). Multiple sequence alignments and phylogenetic relationship analyses revealed DoSAMDC1 had a higher level of sequence similarity to monocot SAMDCs than those of dicot. Expression patterns using qRT-PCR analyses showed that DoSAMDC1 transcripts were expressed constitutively without significant change in the five tissues (not infected with fungi). While in the symbiotic germinated seeds, the expression level was enhanced by 2.74 fold over that in the none-germinated seeds, indicating possible involvement of the gene in symbiotic seed germination of D. officinale.
Adenosylmethionine Decarboxylase ; genetics ; isolation & purification ; Amino Acid Sequence ; Basidiomycota ; physiology ; Cloning, Molecular ; DNA, Complementary ; genetics ; Dendrobium ; enzymology ; genetics ; microbiology ; Germination ; Open Reading Frames ; Phylogeny ; Plants, Medicinal ; enzymology ; genetics ; microbiology ; Seeds ; genetics ; growth & development ; microbiology ; Sequence Alignment ; Symbiosis ; physiology

OBJECTIVETo study the inhibitory effects of antisense bicistronic recombinant adenovirus vector of ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (Ad-ODC-AdoMetDCas) on polyamine biosynthesis,proliferation and invasion of lung cancer cells.

METHODSAdenovirus-mediated gene transduction efficiency was assessed with counting GFP-positive cells using trypan blue. Western Blot and HPLC were used to detect ODC and S-AdoMetDC expression and polyamine content in A-549 cells respectively. Viable cell counting and cell cycle analysis were adopted to evaluate cell growth and cell cycle distribution, and A-549 cell invasion in vitro was detected with Matrigel invasion assay.

RESULTSApproximate 75% of A-549 cells were infected with Ad-ODC-AdoMetDCas when multiplicity of infection reached 50. Our study demonstrated that Ad-ODC-AdoMetDCas vector-mediated gene transfer inhibited tumor cell growth through the blockade of polyamine synthesis pathway. The tumor cells were arrested at cell cycle G1 phase after gene transfer. Gene transferred tumor cells were shown to possess markedly decreased invasiveness.

CONCLUSIONAd-ODC-AdoMetDCas has significant inhibitory effects on lung cancer cell proliferation and invasion and bears therapeutic potential for the treatment of lung cancer.

Adenosylmethionine Decarboxylase ; genetics ; metabolism ; Adenoviridae ; genetics ; Blotting, Western ; Cell Cycle ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Chromatography, High Pressure Liquid ; Genetic Vectors ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Lung Neoplasms ; genetics ; metabolism ; pathology ; Ornithine Decarboxylase ; genetics ; metabolism ; Polyamines ; metabolism ; RNA, Antisense ; genetics ; Transfection