Sebastiano Gattoni-Celli

Effects of 5-azacytidine on methylation and expression of specific dna sequences in c3h 10t1/2 cells.

The present study indicates that the transient exposure of C3H 10T1/2 mouse embryo fibroblasts to 5-azacytidine leads to extensive loss of methylation of the protooncogene c-mos and the beta-globin locus at the cell population level and in at least 40 isolated subclones. These changes persisted, even when the cells were serially passaged for many generations without further exposure to the drug. Even though the amount of demethylation, assessed through differential digestion by the restriction enzymes HpaII and MspI, was quite extensive, neither locus was transcribed at a detectable level. This nonselective analysis suggests, therefore, that loss of DNA methylation is not sufficient per se to induce the expression of certain loci. Presumably, the expression of these loci requires additional factors, some of which may be related to cell lineage and differentiation.

Effects of 5-azacytidine on the progressive nature of cell transformation.

C3H 10T1/2 mouse embryo fibroblasts were exposed to 3 microM 5-azacytidine for 24 h and then serially passaged in the absence of 5-azacytidine and examined for subsequent changes in growth properties. The treated cells showed changes in morphology, saturation density, growth rate, and serum dependence. By the 5th passage they acquired the ability to grow in 0.3% agarose, and by the 30th passage they gave rise to fully transformed foci that grew in agarose, in agar, and in liquid suspension. This progression was rapidly accelerated if the cultures derived from 5-azacytidine-treated cells were exposed for 48 h to the carcinogen benzo[a]pyrene. Results of these studies provide evidence that aberrations in DNA methylation may be one of a series of critical events during the course of multistage carcinogenesis and thus enhance the evolution of tumor cells.

Semiallogeneic cell hybrids as therapeutic vaccines for cancer.

The authors have engineered a cell line that can be used in human studies as a universal donor cell for the formation of semiallogeneic cell hybrids after fusion with patient-derived tumor cells. These hybrids can be irradiated and injected as a patient-tailored therapeutic vaccine in patients affected by virtually any type of cancer. A crucial step in this research effort has been the derivation of an allogeneic cell line (FO1-12) that expresses both a dominant selectable marker (neomycin resistance) and a recessive selectable marker (sensitivity to hypoxanthine, aminopterin, and thymidine), which allows easy selection of semiallogeneic cell hybrids derived from the fusion of FO1-12 cells with patient-derived tumor cells. Tumor-infiltrating lymphocytes derived from select patients with melanoma and exposed to semiallogeneic cell hybrids from the same patient were better able to specifically lyse autologous tumor cells. Furthermore, FO1-12 cells express carcinoembryonic antigen, which is ubiquitous in adenocarcinomas, and fusion of FO1-12 cells with various patient-derived adenocarcinoma cells showed that the hybrid cells also express carcinoembryonic antigen. Because of the results of these preclinical studies, the authors were given permission to use semiallogeneic cell hybrids for immunotherapy of patients with metastatic melanoma or metastatic adenocarcinoma who had not responded to standard treatment regimens. Treatment with semiallogeneic vaccines is associated with minimal or no toxicity and can induce a specific anti-tumor immune response.

Semiallogeneic cancer vaccines formulated with granulocyte-macrophage colony-stimulating factor for patients with metastatic gastrointestinal adenocarcinomas: a pilot phase I study.

The authors report the results of a phase I clinical study using semiallogeneic cancer vaccines formulated with granulocyte-macrophage colony-stimulating factor (GM-CSF) to treat patients with metastatic adenocarcinomas of the gastrointestinal tract. A specially engineered cell line, FO1-12, was used to generate semiallogeneic hybrids by fusion with patient-derived tumor cells; the hybrids express HLA class I and II haplotypes derived from both parental cells. For treatment, the vaccine was mixed with GM-CSF, irradiated, and injected intradermally into patients at weekly or biweekly intervals. Vaccinations were associated with minimal or no toxicity and showed that semiallogeneic hybrids formulated with GM-CSF can induce a specific antitumor immune response in some patients, as measured by a delayed-type hypersensitivity response to autologous tumor cells. Because of the simplicity, feasibility, and flexibility of this immunotherapeutic approach, semiallogeneic hybrid vaccines have the potential to be used in the treatment of virtually any type of cancer.

Semi-allogeneic cell hybrids stimulate HIV-1 envelope-specific cytotoxic T lymphocytes.

ObjectiveThe present study was designed to determine whether the HLA allogeneic T helper response stimulated by semi-allogeneic cell lines could be used as an in vitro model of immune-based therapy to stimulate HIV-specific cytotoxic T lymphocytes. Design and methodsSemi-allogeneic cell hybrids were obtained by the fusion of peripheral blood mononuclear cells from HIV-infected patients with the allogeneic β2-microglobulin-deficient FO1-12 melanoma cell line. These hybrids were used as antigen presenting cells for HIV envelope peptide (env)-specific cytotoxic assays. ResultsThe hybrid cell lines express HLA class I and II antigens from both parental cells, as well as the CD86 costimulatory molecule. HIV-specific cytotoxic T lymphocyte activity was obtained when patients’ peripheral blood mononuclear cells were costimulated with env peptides plus semi-allogeneic hybrids, in contrast with stimulation with either env or hybrid cells alone. Thus, the semi-allogeneic hybrids enhanced HIV-specific killing of target cells. ConclusionsIrradiated, semi-allogeneic cell hybrids engineered for individual AIDS patients provide efficient and simultaneous co-recognition of HLA allogeneic determinants and viral antigenic determinants presented by self-HLA molecules on the same antigen presenting cells and results in the generation of enhanced HIV-specific cytotoxic T lymphocyte activity.

Semi-allogeneic vaccines for patients with cancer and AIDS.

Interest in semi-allogeneic vaccines has been increasing, as demonstrated by the publication of successful preclinical and clinical studies by others and us that validate this immunotherapeutic approach to cancer and HIV-infection. We now report that lymphocytes from an HLA-A2+ melanoma patient, stimulated with a GP100-derived epitope and semi-allogeneic hybrids, lysed target cells presenting this peptide more efficiently than lymphocytes stimulated with GP100 peptide alone. Phenotypic analysis with GP100/HLA-A2 tetramer complexes also demonstrated a significant increase in the size of the GP100-specific CD8+ lymphocyte pool when PBMC were stimulated with both peptide and semi-allogeneic hybrids. Analyses of PBMC from other donors further suggest that this stimulatory effect of semi-allogeneic hybrids results from an increase in the HLA-restricted recall response of CD8+ cytotoxic T lymphocytes against melanoma-derived antigens. Likewise, lymphocytes from an HIV-infected patient that had been stimulated with a mixture of HIV-derived peptides and semi-allogeneic hybrids also demonstrated significantly greater antigen-specific cytotoxicity and tetramer reactivity compared with those lymphocytes that had been stimulated with peptides alone. Our approach should provide useful information for the design of future clinical studies treating patients with melanoma or HIV with therapeutic vaccines consisting of a combination of semi-allogeneic hybrids and immunodominant antigenic peptides.