When human U937 cells are placed in agarose microbeads and treated with a detergent, the cytoplasmic membrane is lysed and the nuclear membrane is permeabilized. However, the nuclei remain intact and maintain both replication and transcription. Biotin labeled monoclonal antibodies against Z‐DNA have been diffused into this system and used to measure the amount of Z‐DNA present in the nuclei. It has previously been shown that the amount of Z‐DNA present decreases due to relaxation by topoisomerase I and increases as the level of transcription increases. Here we measure the formation of Z‐DNA in the c‐myc gene by crosslinking the antibodies to DNA using laser radiation at 266 nm for 10 ns. The crosslinked DNA is isolated by restriction digestion, separation of antibody labeled fractions through the biotin residue, and subsequent proteolysis to remove the crosslinked antibody. Three AluI restriction fragments of the c‐myc gene are shown to form Z‐DNA when the cell is transcribing c‐myc. The Z‐DNA forming segments are near the promoter regions of the gene. However, when U937 cells start to differentiate and transcription of the c‐myc gene is down‐regulated, the Z‐DNA content goes to undetectable levels within 30–60 min.

Summary Natural killer-like T lymphocytes termed cytokine-induced killer (CIK) cells have been shown to eradicate established tumours in a severe combined immune deficient (SCID) mouse/human lymphoma model. Recently, we demonstrated that CIK cells transfected with cytokine genes possess an improved proliferation rate and a significantly higher cytotoxic activity as compared to non-transfected cells. Here, in a phase I clinical protocol, autologous CIK cells were generated from peripheral blood obtained by leukapheresis in patients with metastatic renal cell carcinoma, colorectal carcinoma and lymphoma. CIK cells were transfected with a plasmid containing the interleukin-2 (IL-2) gene via electroporation. Transfected cells generated IL-2 in the range of 330-1800 pg 10 -6 cells 24 h -1 with a mean of 836 pg 10 -6 cells 24 h -1. Ten patients received 1-5 intravenous infusions of IL-2-transfected CIK cells; five infusions with transfected CIK cells were given. In addition, the same patients received five infusions with untransfected CIK cells for control reasons. In three patients, WHO grade 2 fever was observed. Based on polymerase chain reaction of peripheral blood transfected cells could be detected for up to 2 weeks after infusion. There was a significant increase in serum levels of interferon gamma (IFN-γ), granulocyte-macrophage colony-stimulating factor (GM-CSF) and transforming growth factor beta (TGF-β) during treatment. Interestingly, there was also an increase in CD3+ lymphocytes in the blood of patients during therapy. In accordance, a partial increase in cytotoxic activity in peripheral blood lymphocytes (PBLs) was documented when patient samples before and after therapy were compared. Concerning clinical outcome, six patients remained in progressive disease, three patients showed no change by treatment, and one patient with lymphoma developed a complete response. In conclusion, we were able to demonstrate that CIK cells transfected with the IL-2 gene can be administered without major side-effects and are promising for future therapeutic trials.

Mammalian cells have been encapsulated in agarose microbeads, and from these cells metabolically active permeabilized nuclei were prepared. Previously, we showed that biotin-labeled monoclonal antibodies against Z-DNA can be diffused into the nuclei and, over a specific concentration range, they will bind to Z-DNA within the nucleus in a concentration-independent manner. By using radiolabeled streptavidin, we showed that the amount of Z-DNA antibody bound is related to the torsional strain of the DNA in the nucleus. Relaxation of the DNA results in a decrease of Z-DNA formation, whereas increasing torsional strain through inhibiting topoisomerase I results in increased Z-DNA formation. Here we measure the influence of RNA transcription and DNA replication. Transcription is associated with a substantial increase in the binding of anti-Z-DNA antibodies, paralleling the increased level of RNA synthesized as the level of ribonudleoside triphosphate in the medium is increased. DNA replication yields smaller increases in the binding of Z-DNA antibodies. Stopping RNA transcription with inhibitors results in a large loss of Z-DNA antibody binding, whereas only a small decrease is associated with inhibition of DNA replication.Z-DNA exists in biological systems in a dynamic rather than a static state. B-DNA is the lower-energy conformation and to put DNA into the left-handed Z conformation, energy has to be expended. In the cell nucleus, DNA is found at various levels of unwinding or negative supercoiling. Negative supercoiling is a major factor in stabilizing left-handed Z-DNA (1-5). Through the action of topoisomerases, DNA loses negative supercoiling and the left-handed Z state flips back to the lower-energy right-handed B-DNA state. The experiments reported herein must be viewed in the context of an equilibrium between B-DNA and Z-DNA, which in the face of topoisomerases requires the input of a steady process to maintain the Z conformation.A system has been developed for measuring the level of Z-DNA in metabolically active eukaryotic nuclei (6). Nuclear preparations developed by Jackson (7) and Jackson and Cook (8-10) are used in which living mammalian cells trapped in agarose microbeads maintain normal metabolism. Treating the cells with 0.5% Triton X-100 results in lysis of the cytoplasmic membrane and permeabilization of the nuclear membrane. The nucleus remains morphologically intact and these nuclei are metabolically active (7-10). They carry out DNA replication at a rate 85% that seen in the intact cell and maintain transcription. The nuclei are permeable to macromolecules and we have used (6) this system to measure the binding of monoclonal antibodies that bind to Z-DNA independent of the nucleotide sequence. A single biotin residue is conjugated to the monoclonal antibody so that the binding can be measured quantitatively by adding radiolabeled streptavidin. At triphosphates (dNTPs) to measure the effect of transcription or replication on the binding of antibodies against Z-DNA. We find that transc...

Cytokine gene transfer into tumor cells has been shown subsequent first clinical phase I study six patients with to mediate tumor regression and antimetastatic effects in metastatic melanoma were vaccinated with autologous, several animal models via immunomodulation. Therefore, interleukin-12 gene-modified tumor cells. Melanoma cells clinical protocols have been developed to treat cancer were expanded in vitro from surgically removed metastpatients with cytokine gene-modified tumor cells. We ases, transduced by ballistic gene transfer, irradiated and inserted the genes coding for the p35 and p40 chain of were then injected subcutaneously (s.c.) at weekly interinterleukin-12 (IL-12) in two independent eukaryotic vals. Clinically, there was no major toxicity except for mild expression vectors and transduced melanoma cells of 15 fever. All patients completed more than four s.c. vaccidifferent primary tumor cultures with both plasmids by a nations over 6 weeks and were eligible for immunological ballistic gene transfer approach. Secreted IL-12 demonevaluation. Post-vaccination, peripheral mononuclear cells strated strong bioactivity by inducing interferon-␥ release were found to contain an increased number of tumorfrom peripheral blood lymphocytes upon coculture with cell reactive proliferative as well as cytolytic cells as determculture supernatants after IL-12 gene transfer which could ined by a limiting dilution analysis in two patients. Two at least partly be blocked by IL-12-specific antisera. Further patients developed DTH reactivity against autologous enrichment of transduced tumor cells by magnetic separmelanoma cells and one had a minor clinical response. ation directly after gene transfer increased cytokine Biopsies taken from that patient's metastases revealed a secretion from a mean of 119 pg in the unsorted to 507 heavy infiltration of CD4 + and CD8 + T lymphocytes. In conpg IL-12 (24 h/10 6 cells) in the magnetically enriched cell clusion, vaccination induced immunological changes even fraction. Irradiation of these cells led to a further elevation in a group of advanced, terminally ill patients. These of secreted IL-12 (mean 987 pg). Elevated IL-12 levels changes can be interpreted as an increased antitumor were detected over 7 days after irradiation in vitro. In a immune response.

Abstract. Permeabilized nuclei from mammalian cells encapsulated within agarose microbeads in an isotonic buffer are active in transcription and replication (Jackson, D. A., and P. R. Cook. 1985. . Their DNA is intact and the nuclei are accessible to macromolecules. Myeloma nuclei prepared in this way were used to probe the extent of DNA negative supercoiling and the effects of altering torsional strain by binding radioactively labeled monoclonal antibodies to Z-DNA. Control experiments used monoclonal antibodies against a nonhistone chromosomal protein, HMG-17. On increasing the amount of anti-HMG-17 added, a binding plateau was reached encompassing a 200-fold range of antibody concentration. On binding anti-Z-DNA antibody, a similar broad plateau of constant binding was found encompassing a 100-fold range of antibody concentration. The latter result was taken as a measure of preexisting Z-DNA in the nuclei. Additional anti-Z-DNA antibody binding can be "induced" in the presence of much higher concentration of antibody, apparently by perturbing the B-DNA/Z-DNA equilibrium. On inhibiting topoisomerase I with camptothecin, an elevated antibody binding plateau was found, suggesting that elastic torsional strain in the DNA is responsible for stabilizing the preexisting Z-DNA. This interpretation is supported by the fact that addition of small, nicking amounts of DNase I leads to a complete loss of antibody binding in the Z-DNA plateau region but not in the region of "induced" Z-DNA.