RESUMO
Background:Deoxyhypusine synthase(DHPS)is a key factor in post-translational modification of the precursor of eukaryotic initiation factor 5A(eIF-5A),and eIF-5A is closely related to the regulation of proliferation and invasion of tumor cells. Aims:To investigate the expression of DHPS in gastric cancer and its clinical significance,and to explore the possible mechanism of its effect on metastasis of gastric cancer. Methods:Tissue microarray containing 92 gastric cancer tissues and paired adjacent cancerous tissues was employed to detect the DHPS expression by using immunohistochemical staining,and the correlation of DHPS expression with the clinicopathological characteristics of gastric cancer was analyzed. DHPS-siRNA and GC7,an inhibitor of DHPS were used,respectively to intervene human gastric cancer cell line MGC803. The invasive ability of MGC803 cells was assessed by cell invasion assay,and the expressions of metastasis-related proteins including vascular endothelial growth factor(VEGF),matrix metalloproteinase 2(MMP2)and MMP9 were detected by ELISA. Results:In 62(67.4%)cases of gastric cancer,DHPS was highly expressed,and its expression was closely related to tumor diameter,TNM stage and depth of invasion(P <0.05),but not related to gender,age,lymph node metastasis and distant metastasis(P >0. 05). Both DHPS-siRNA and GC7 could down-regulate the invasiveness of MGC803 cells,while the former could also reduce the expressions of VEGF,MMP2 and MMP9 proteins(P <0.05). Conclusions:DHPS is highly expressed in gastric cancer and associated with tumor invasion and progression. DHPS is expected to be a new target for diagnosis and treatment of gastric cancer because of its regulatory effect on invasion and metastasis of tumor cells.
RESUMO
The biosynthesis of hypusine [Nepsilon-(4-amino-2-hydroxybutyl)-lysine] occurs in the eIF-5A precursor protein through two step posttranslational modification involving deoxyhypusine synthase which catalyzes transfer of the butylamine moiety of spermidine to the epsilon-amino group of a designated lysine residue and subsequent hydroxylation of this intermediate. This enzyme is exclusively required for cell viability and growth of yeast (Park, M.H. et al., J. Biol. Chem. 273: 1677-1683, 1998). In an effort to understand structure-function relationship of deoxyhypusine synthase, posttranslational modification(s) of the enzyme by protein kinases were carried out for a possible cellular modulation of this enzyme. And also twelve deletion mutants were constructed, expressed in E. coli system, and enzyme activities were examined. The results showed that deoxyhypusine synthase was phosphorylated by PKC in vitro but not by p56lck and p60c-src. Treatment with PMA specifically increased the relative phosphorylation of the enzyme supporting PKC was involved. Phosphoamino acid analysis of this enzyme revealed that deoxyhypusine synthase is mostly phosphorylated on serine residue and weakly on threonine. Removal of Met1-Glu10 (deltaMet1-Glu10) residues from amino terminal showed no effect on the catalytic activity but further deletion (deltaMet1-Ser20) caused loss of enzyme activity. The enzyme with internal deletion, deltaGln197-Asn212 (residues not present in the human enzyme) was found to be inactive. Removal of 5 residues from carboxyl terminal, deltaLys383-Asn387, retained only slight activity. These results suggested that deoxyhypusine synthase is substrate for PKC dependent phosphorylation and requires most of the polypeptide chains for enzyme activity except the first 15 residues of N-terminal despite of N- and C-terminal residues of the enzyme consist of variable regions. Copyright 2000 Academic Press.