ABSTRACT
the murine urothelial cell line, mb49 was transfected with the reporter gene pcmvlacz using a number of commercial transfection agents. the transfection efficiency of these agents, as determined by beta-galactosidase activity, is in the order of dotap>superfect>Fugene. The addition of methyl-beta-cyclodextrin solubilized cholesterol (MBC) to Dotap and Superfect further improved their transfection efficiency by 3.8-fold and 2.6-fold, respectively. beta-Galactosidase activity was detectable within 1 h of transfection and peaked at 48 h. Nuclear and cytoplasmic separation showed that with Dotap + methyl-beta-cyclodextrin solubilized cholesterol (DMBC), the DNA plasmid complex was found in both the nucleus and the cytoplasm. In vivo, murine bladders were transfected with an intravesical instillation of DMBC + DNA for 2 h. Two days later the bladder, lungs, liver, spleen and heart were assayed for the presence of the beta-galactosidase gene by staining and PCR. Expression of the gene was confined to the bladder. Both in vitro and in vivo expression was observed after as little as a 15 min exposure to DMBC:DNA. Expression of the marker gene was present up to 30 days after transfection in vivo. From our data it appears that DMBC is the best nonviral agent for the transfection of urothelial cells in vitro and in vivo.
Subject(s)
Genetic Vectors/administration & dosage , Transfection/methods , Tumor Cells, Cultured , Urothelium , Animals , Cell Nucleus/enzymology , Cholesterol , Cytoplasm/enzymology , Fatty Acids, Monounsaturated , Female , Gene Expression , Lipids , Mice , Mice, Inbred C57BL , Quaternary Ammonium Compounds , Time Factors , Urinary Bladder , beta-Galactosidase/geneticsABSTRACT
Bladder wash-derived lymphocytes from superficial bladder cancer patients involved in high dose BCG, low dose BCG, and low dose BCG with IFN-alpha treatments were examined. We found an increasing trend in the percentage of CD3 T cells with each weekly intravesical instillation and the proportion of CD3 T cells expressing the gammadelta T cell receptor was significantly higher in patients receiving standard dose BCG than those receiving low dose BCG or low dose BCG plus IFN-alpha. Most patients had a predominance of CD4 T cells, while some had more CD8 T cells. The CD4/CD8 ratio did not vary much during the instillations. Surprisingly, both patients and normal control individuals had high percentages of CD69- and CD45RO-positive lymphocytes in the bladder wash and this was not reflected in lymphocytes from peripheral blood collected in parallel. We found no differences in lymphocyte phenotypes, cytokine production, and clinical outcome in the patients from three arms. This may reflect that the qualitative and quantitative immune responses elicited from the three treatments are similar. However, the lymphokine-activated killing ability of peripheral blood lymphocytes against allogeneic cell-lines from the cancer patients was depressed compared to normal individuals and the cytotoxicity appeared to be enhanced after intravesical treatment.
