The apoptosis-inducing activity of human selenoprotein P shorter isoform / 中华肿瘤杂志

<p><b>OBJECTIVE</b>Human selenoprotein P (HSelP) is unique protein that contains 10 selenocysteines encoded by 10 inframe UGA, which typically function as stop codon. The function of HSelP remains unclear, in part due to the inability to express it by gene recombinant technique. This study is to investigate expression and purification of recombinant HSelP in prokaryotic expression system, and its activity to induce apoptosis in vitro.</p><p><b>METHODS</b>The shorter HSelP isoform was cloned. After the selenocysteine (SeCys) at 40th position from N terminus of the HSelP shorter isoform was mutated into cysteine by PCR, it was expressed in E. coli. The expressed product was purified with DEAE column and identified by Western blot. Subsequently, its function on induction of mitochondrial apoptotic activity was studied.</p><p><b>RESULTS</b>The mutant HSelP shorter isoform expressed in prokaryotic system was purified by DEAE column to 90% homogeneity. The purified product, HSelP280m, induced the opening of mitochondrial permeability transition pore (PTP) and decreased the transmembrane potential in a dose-dependent manner. These events could be abolished by PTP specific inhibitors.</p><p><b>CONCLUSION</b>HSelP280m can induce the opening of mitochondrial PTP, which provides a basis for investigating the structure and function of recombinant HSelP.</p>

Antitumor effect of gene gun-mediated DNA vaccine pWRG-neu immunization in C57BL/6 mice / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the inhibitory effect of DNA vaccine immunization on neu-overexpressed melanoma growth in prophylactic treatment and anti-lung-metastasis experiments in C57BL/6 mice.</p><p><b>METHODS</b>pcDNA-neu transfected into B16F10 with transfection reagent Fugene 6, neu-overexpressed cell clone B16F10-neu was selected with limited dilution method. The growth curve was drawn to analyse its proliferating character in vitro. With Helios gene gun system, DNA vaccine pWRG-neu was immunized to 8-week-old C57BL/6 mice in the shaved abdominal skin for 3 times at two-weekly interval. After immunization, the life span was analyzed. Using MTT assay, the cytolysis activity of the DNA immunized mice spleen cells was compared.</p><p><b>RESULTS</b>One clone of neu-overexpressed B16F10-neu was selected and its proliferating character was the same as B16F10 and B16F10-pcDNA. In prophylactic, treatment and anti-lung-metastasis experiments, gene gun-mediated pWRG-neu immunization could exhibit antitumor effects. The growth and metastasis of neu-overexpressed melanoma was reduced dramatically. The spleen cells of the immuned mice showed cytotoxic T lymphocyte (CTL) activity.</p><p><b>CONCLUSION</b>Gene gun-mediated gene transfer is effective and practicable. DNA vaccine pWRG-neu is potent in preventing subsequent tumor cells challenge, inhibiting the tumor growth and metastasis.</p>