Transfection of primary tumor cells and tumor cell lines with plasmid DNA/lipid complexes.

Cancer vaccines that utilize genetically modified tumor cells require gene transfer methods capable of producing immunostimulatory doses of transgenes from fresh or short-term cultures of human tumor cells. Our studies optimize in vitro transfection of primary tumor cells using cationic lipids and a plasmid encoding the gene for human interleukin-2 (IL-2). Established tumor cell lines produced 10- to 100-fold more IL-2 than did fresh or short-term tumor cultures as measured by enzyme-linked immunoabsorbent analysis. Importantly, transfection of primary tumor cells produced immunostimulatory levels of IL-2 as determined by increased thymidine incorporation by autologous peripheral blood mononuclear cells and lymphokine-activated killer cell activity. IL-2 secretion by tumor cells persisted for at least 30 days post-transfection and was unaffected by freeze thawing or irradiation to 8000 rads. Multiple solid tumor types were successfully transfected, but normal blood mononuclear cells and leukemic blasts were resistant to transfection. Enzyme-linked immunoabsorbent analysis of the amount of IL-2 secreted into the medium by transfected tumor cells correlated with the percentage of tumor cells expressing intracellular IL-2 as measured by flow cytometry. Plasmids utilizing a cytomegalovirus promoter yielded superior transfection efficiencies compared with plasmids containing a Rous sarcoma virus promoter. These results suggest that a clinical vaccine trial using autologous tumor cells genetically modified to secrete IL-2 is feasible in patients with solid tumors.

Polycations and cationic lipids enhance adenovirus transduction and transgene expression in tumor cells.

Replication-deficient adenovirus vectors are efficient vehicles for delivering therapeutic genes into mammalian cells. However, the high doses required to produce effective gene transfer in vivo can also cause unwanted cellular toxicity. To improve replication-deficient adenovirus transgene expression while minimizing adverse reactions, we have tested polycationic compounds for their ability to enhance adenovirus adsorption. We demonstrate increased transgene expression after mixing adenovirus preparations with polycations, cationic lipids, and CaCl2 prior to transduction in vitro. An E1-deleted adenovirus vector was admixed with various polycations, and beta-galactosidase (beta-gal) activity was evaluated. The optimal polycation concentrations for augmenting adenovirus-mediated gene transfer were 5-10 microg/mL polybrene, 400 microg/mL protamine sulfate, 10 microg/mL N-(1-[2,3-dioleoyloxy]propyl)-N,N,N-trimethylammonium methylsulfate (DOTAP), 2.5 microg/mL Lipofectamine, and 62.5 mM CaCl2. Polycations enhanced beta-gal expression in three of six established cell lines. Similar results were obtained using primary tumor cell cultures, where beta-gal expression was increased 1.5- to 10.7-fold (mean = 3.6) by polybrene, 1.8- to 7.5-fold (mean = 3.4) by DOTAP, and 2.3- to 10.4-fold (mean = 4.8) by protamine sulfate. Adenovirus transduction efficiency in two primary leukemia isolates was improved by 3- and 4.5-fold. We were unable to demonstrate any benefit when adenovirus was admixed with protamine sulfate prior to intratumoral injection in a xenogeneic severe combined immunodeficient mouse melanoma tumor model. Further studies will determine whether polycations can improve intratumoral gene transfer.

Construction of new amplifier expression vectors for high levels of IL-2 gene expression.

The success of IL-2 gene therapy in cancer is in part dependent on the development of high level IL-2 gene expression vectors. Currently, expression vectors based on the human cytomegalovirus (CMV) promoter give the highest levels of expression. We have attempted to construct new IL-2 expression vectors to test whether gene expression can be further increased. The first approach was to use the new SR-alpha promoter to control IL-2 gene expression. The second approach was to combine the Tat transcription activator gene and the HIV 1 and 2 promoters in the same construct so that the levels of gene expression can be amplified. Transient transfection results using the human colon cancer cell line SW480 showed that the SR-alpha promoter yields similar levels of activity as the CMV promoter. However, the HIV 1 and 2 promoter-based amplifier constructs produced 11 and 28 times more secreted IL-2 than the CMV promoter control. The augmented activity of the amplifier constructs was dependent on the presence of the Tat gene and the transcriptional units must be placed in the same orientation. Reducing the size of the vectors by elimination of the neomycin selectable marker did not increase the activity of the constructs.

SEM-dissection of a human embryo derived from an ectopic pregnancy.

A 6.5-week-old human embryo with an approximate crown-rump length of 13.2 mm was obtained from a tubal pregnancy. Two hours before surgical removal, the embryo was imaged with real time ultrasound and was noted to have rhythmic cardiac motion. Subsequent to surgical removal, the embryo was dissected free from the placenta and prepared for routine scanning electron microscopic (SEM) studies. Progressive stages of dissection with microsurgical instruments followed by SEM photography elucidated the three-dimensional aspects of embryonic development of many structures, including the lens placode, tongue bud, Rathke's pouch, atrial and ventricular foramina, primitive intestinal loop and undifferentiated external genitalia. Almost certainly, such clear views of dissected structures can contribute to our understanding of human embryonic development.

Cytostatic and cytotoxic activity of lymphokine-activated killer cell supernatants.

Incubation of peripheral blood mononuclear cells with interleukin-2 (IL-2) results in the release of a factor which is cytostatic and cytotoxic both to tumor cell lines (A375M, A375P, C480, MCF-7, Hey) and fresh tumor cells (in the human tumor cloning assay), including breast cancer, colon cancer, melanoma, myeloma and ovarian cancer. The factor cannot be detected in a 4-h chromium-release assay, but is best demonstrated after tumor cells have been to it for exposed 3 days. The factor is not cytotoxic to normal peripheral blood leukocytes or normal fibroblasts, and is not toxic to certain targets sensitive to lymphokine-activated killer (LAK) cells, such as K562 and Daudi cells. The factor is diffusible, non-dialyzable, relatively stable to heat and acid and does not contain appreciable amounts of targets resistant to interferon-alpha and beta, tumor necrosis factor beta and interleukin-1. The data suggest that there are several mechanisms of LAK cell activity against tumor cells including one which requires direct interaction of LAK and tumor cells and one which is mediated by LAK cell supernatant. The former is detected by 4-h chromium release while the latter is not.