Counterselection and co-delivery of transposon and transposase functions for Sleeping Beauty-mediated transposition in cultured mammalian cells.

Sleeping Beauty (SB) is a gene-insertion system reconstructed from transposon sequences found in teleost fish and is capable of mediating the transposition of DNA sequences from transfected plasmids into the chromosomes of vertebrate cell populations. The SB system consists of a transposon, made up of a gene of interest flanked by transposon inverted repeats, and a source of transposase. Here we carried out a series of studies to further characterize SB-mediated transposition as a tool for gene transfer to chromosomes and ultimately for human gene therapy. Transfection of mouse 3T3 cells, HeLa cells, and human A549 lung carcinoma cells with a transposon containing the neomycin phosphotransferase (NEO) gene resulted in a several-fold increase in drug-resistant colony formation when co-transfected with a plasmid expressing the SB transposase. A transposon containing a methotrexate-resistant dihydrofolate reductase gene was also found to confer an increased frequency of methotrexate-resistant colony formation when co-transfected with SB transposase-encoding plasmid. A plasmid containing a herpes simplex virus thymidine kinase gene as well as a transposon containing a NEO gene was used for counterselection against random recombinants (NEO+TK+) in medium containing G418 plus ganciclovir. Effective counterselection required a recovery period of 5 days after transfection before shifting into medium containing ganciclovir to allow time for transiently expressed thymidine kinase activity to subside in cells not stably transfected. Southern analysis of clonal isolates indicated a shift from random recombination events toward transposition events when clones were isolated in medium containing ganciclovir as well as G418. We found that including both transposon and transposase functions on the same plasmid substantially increased the stable gene transfer frequency in Huh7 human hepatoma cells. The results from these experiments contribute technical and conceptual insight into the process of transposition in mammalian cells, and into the optimal provision of transposon and transposase functions that may be applicable to gene therapy studies.

Somatostatin receptor gene transfer inhibits established pancreatic cancer xenografts.

BACKGROUND: Most human pancreatic adenocarcinoma cells do not express somatostatin receptors, and somatostatin does not inhibit the growth of these cancers. We have demonstrated previously that somatostatin inhibits the growth of pancreatic cancers expressing somatostatin receptor subtype-2 (SSTR2), but not receptor-negative cancers. SSTR2 expression may be an important tumor-suppressor pathway that is lost in human pancreatic cancer. We hypothesized that SSTR2 gene transfer would restore the growth-inhibitory response of human pancreatic cancer to somatostatin. MATERIALS AND METHODS: Palpable human pancreatic adenocarcinoma tumors were established on the backs of nude mice by subcutaneous injection of cultured cells (Panc-1). The animals were divided into 5 groups (n = 10/group). Group I served as an untreated control. Group II received an intramuscular injection of the long-acting somatostatin analogue Sandostatin LAR. Group III received Lac-Z expressing adenovirus via intraperitoneal injection. Group IV received SSTR2 expressing adenovirus via intraperitoneal injection. Group V received SSTR2 expressing adenovirus via intraperitoneal injection and an intramuscular injection of Sandostatin LAR. The rate of tumor growth was assessed with calipers. After 28 days, the animals were anesthetized and exsanguanated, and the tumors were excised and weighed. Plasma somatostatin and octreotide levels were measured by radioimmunoassay. Expression of cell-surface somatostatin-receptor protein and known tumor-suppressor proteins was determined by reverse transcriptase-polymerase chain reaction, Western blot, and immunohistochemistry. RESULTS: Systemic delivery of SSTR2-expressing adenovirus by intraperitoneal injection resulted in expression of SSTR2 protein in the subcutaneous human pancreatic cancers. Final tumor weight was significantly decreased in the groups expressing SSTR2 receptors compared to the other 3 groups. Treatment with Sandostatin LAR increased plasma octreotide levels as determined by radioimmunoassay, but had no significant effect on tumor growth. Western blot analysis revealed an up-regulation of the cyclin-dependent kinase inhibitors p27 and p16 in the SSTR2 transfected tumors. CONCLUSIONS: Expression of SSTR2 by human pancreatic cancer causes significant slowing of tumor growth by a mechanism independent of exogenous somatostatin. The mechanism may involve up-regulation of known tumor-suppressor proteins. Restoration of SSTR2 gene expression deserves further study as a potential gene-therapy strategy in human pancreatic cancer.

Somatostatin receptor subtype 2 gene therapy inhibits pancreatic cancer in vitro.

BACKGROUND: Most human pancreatic adenocarcinoma cells do not express somatostatin receptors and somatostatin does not inhibit the growth of these cancers. We have demonstrated previously that somatostatin inhibits the growth of pancreatic cancers expressing somatostatin receptor subtype 2 (SSR2) but not receptor-negative cancers. SSR2 expression may be an important tumor suppressor pathway that is lost in human pancreatic cancer. We hypothesized that SSR2 gene transfer would restore the growth inhibitory response of human pancreatic cancer to somatostatin. METHODS: We created adenoviral constructs containing the SSR2 or Lac-Z gene and transfected somatostatin receptor-negative human pancreatic cancer cells (Panc-1). Presence of functional cell surface SSR2 protein was assessed by whole-cell competitive binding assays. Parental cells, Lac-Z-transfected, and SSR2-transfected cells were cultured in the presence and absence of somatostatin. The rate of cell growth was determined by direct cell counting using a hemacytometer (n = 8 wells/group). Cells were analyzed for expression of tumor suppressor proteins by Western blot. RESULTS: Panc-1 cells transfected with the SSR2 transgene demonstrated high-affinity specific binding of (125)I-somatostatin at physiologic concentrations. Expression of somatostatin receptors caused 60% inhibition of cell growth compared with the Lac-Z virus-treated controls (P < 0.05 by Kruskal-Wallis/Bonferroni). There was no additional inhibition of cell proliferation with exogenous somatostatin. Furthermore, addition of somatostatin ligand antibody did not diminish the effect of SSR2 expression on cell proliferation. Western blot analysis revealed an upregulation of the cyclin-dependent kinase inhibitor p27 in the SSR2-transfected cells. CONCLUSIONS: Expression of SSR2 by human pancreatic cancer causes significant slowing of cell division by a mechanism independent of somatostatin. The mechanism may involve upregulation of known tumor suppressor proteins. Restoration of SSR2 gene expression deserves further study as a potential gene therapy strategy in human pancreatic cancer.