Biomedical subjects

S Gattoni-Celli

Publications and source records attributed to S Gattoni-Celli.

43 records · Page 3Linked to original sources

PubMed1983

Design of a retrovirus-derived vector for expression and transduction of exogenous genes in mammalian cells.

We have developed a transfection vector for animal cells that contains long terminal repeat (LTR) sequences to promote expression. Plasmid p101/101, a derivative of plasmid pBR322 containing the complete Moloney murine sarcoma virus genome, was cut with restriction enzymes and religated so that both the 5' and 3' LTRs were retained and all but about 700 base pairs of the intervening viral sequences were removed. To test this vector, the Escherichia coli gene gpt was cloned into a unique PstI site, between the two LTRs, with guanine and cytosine tailing, a method that can be generalized for insertion of any DNA segment into this vector. When DNA from recombinant plasmids in which the gpt gene was inserted in the same transcriptional polarity as the LTR sequences was transfected onto murine or rat fibroblast cultures, we obtained a high yield of Gpt(+) colonies. However, plasmid constructs with the gpt gene in the opposite polarity were virtually devoid of activity. With gpt in the proper orientation, restriction enzyme cuts within the LTRs or between the 5' LTR and the gpt gene reduced transfection by more than 98%, whereas a cut between the gpt gene and the 3' LTR gave an 80% reduction in activity. Thus, both 5' and 3' LTR sequences are essential for optimal gpt expression, although the 5' LTR appears to play a more important role. When the LTR-gpt plasmid was transfected onto murine leukemia virus-infected mouse fibroblasts, we obtained evidence that RNA copies became pseudotyped into viral particles which could transfer the Gpt(+) phenotype into rodent cells with extremely high efficiency. This vector should prove useful for high-efficiency transduction of a variety of genes in mammalian cells.

Animals↗

PubMed1983

Molecular mechanisms of tumor promotion and multistage carcinogenesis.

Carcinogenesis is a multistep process resulting from a complex interaction between multiple factors, both environmental and exogenous. In contract to initiating agents that act by damaging cellular DNA, the primary targets of the phorbol ester tumor promoters are membrane-associated receptors. We have proposed a stereochemical model to explain the interaction of these amphiphilic molecules, and of teleocidin and aplysiatoxin, with this receptor system. The model is consistent with evidence that a complex between protein kinase C and phospholipid is the actual receptor for these compounds. Recent data we have obtained with a compound present in tung oil, 12-O-hexadecanoyl-16-hydroxyphorbol-13-acetate (HHPA), and twelve of its congener's (provided by Y. Ito et al.) are also consistent with our stereochemical model. We have studied phorbol ester receptors in a wide variety of tissue culture cell types. Our data, together with other findings, provide evidence for considerable receptor heterogeneity and this may relate to the pleiotropic effects of these compounds. We have found a case of "masked" receptors in a rat liver cell line and shown that it is due to a cell-associated esterase. Normal human melanocyte cultures contain phorbol ester receptors and this is of particular interest since these cells actually require these or related compounds for optimal growth (in collaboration with M. Eisinger). The receptor studies provide clues to how tumor promoters can, via inductive mechanisms, produce alterations in the structure and function of cell membranes. It is not known, however, how in the multistep carcinogenic process promoters enhance the eventual outgrowth of permanently altered tumor cells. We have found that TPA and teleocidin produce a marked enhancement of transformation of C3H 10T1/2 cells induced by transfection with h-ras human bladder cancer oncogene. These and other results are discussed in terms of the role of alterations in cellular oncogenes and transcriptional enhancer sequences during multistage carcinogenesis.

Adenosine Triphosphate↗

PubMed1982

Carcinogen- and radiation-transformed C3H 10T1/2 cells contain RNAs homologous to the long terminal repeat sequence of a murine leukemia virus.

Carcinogen- or radiation-transformed C3H 10T1/2 murine fibroblasts transcribe a set of poly(A)+RNAs that contain sequences homologous to the long terminal repeat (LTR) sequence of Moloney murine sarcoma virus. These LTR-containing RNAs consist of a series of discrete bands ranging in size from about 38 to 18 S. The higher molecular weight molecules (30-38 S) in this set of RNAs also contain sequences homologous to the gag, pol, and env genes of a murine leukemia virus. A 24S RNA contains sequences homologous to the env gene of murine leukemia virus. A 20S and an 18S RNA also share homology with the LTR probe but fail to hybridize to the gag, pol, or env probes or to a probe for the U3 region of the LTR sequence. Thus, the latter transcripts do not appear to arise from a known endogenous murine leukemia virus genome. Although this entire set of RNAs is absent from normal C3H 10T1/2 cells (or is present at an extremely low level), these RNAs are induced by BrdUrd or 5-azacytidine. The presence of these RNAs may provide highly sensitive molecular markers of transformation of murine cells.

Animals↗

PubMed1982

The possible role of cellular genes related to retroviruses in the process of chemical and radiation carcinogenesis.

Chemical carcinogens, radiation, and tumor promoters can not introduce new genetic information into cells. They must, therefore, call upon genes already present in the target cell. We have recently explored the possibility that these host genes share homology with specific nucleic acid sequences present in some of the known retroviruses. In one set of experiments we used a cloned DNA probe prepared from the oncogene (v-mos) of Moloney murine sarcoma virus (Mo-MSV) to examine the state of integration, methylation, and transcription of the homologous cellular oncogene (c-mos) in normal murine cells and in murine cells transformed by radiation or chemical carcinogens. We found that in the transformed cells the c-mos sequence has not undergone rearrangement within the host genome and, as in normal cells, this sequence is hypermethylated and transcriptionally silent. These results are in sharp contrast to the situation in cells transformed by Mo-MSV in which the exogenous v-mos sequence is integrated at new sites within the host genome, is undermethylated and is extensively transcribed. In related studies, we have found that another one gene, c-ras (H), also fails to show any evidence for rearrangement in carcinogen or radiation transformed C3H 10T1/2 cells. Since there is evidence that eukaryotic cells contain numerous onc genes our results do not rule out the possibility that onc genes other than c-mos and c-ras (H) may play a role in the transformation of murine cells by chemicals or radiation. Additional studies utilizing probes to other onc genes are required to evaluate this possibility.

Animals↗

PubMedDate not supplied

Rearranged c-mos locus in a MOPC 21 murine myeloma cell line and its persistence in hybridomas.

Studies of a number of normal and carcinogen-transformed murine cell lines, and a variety of murine tissues, have indicated that, in contrast to several other cellular oncogenes, the oncogene c-mos gene is usually transcriptionally silent. The recent report by Rechavi et al. indicating that in the mouse myeloma XRPC24 originally induced by pristane (2,6,10-14-tetramethylpentadecane) the c-mos gene is rearranged and transcriptionally active, and that it can transform murine fibroblasts in a transfection assay, is therefore of considerable interest. Here we show that the c-mos locus has undergone a similar rearrangement, and is also transcriptionally active, in the cell line P3-X63-Ag8-653, a derivative of the mouse myeloma MOPC 21 which was induced by mineral oil. This line is widely used for making hybridomas that synthesize monoclonal antibodies. We also demonstrate that the rearranged c-mos sequence is maintained in three different hybridomas derived by fusion of this cell line with normal murine spleen lymphocytes, suggesting that it may play a role in the continuous growth and/or constitutive immunoglobulin production by these hybridomas.

Animals↗

PubMedDate not supplied

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.

Adenocarcinoma↗

PubMedDate not supplied

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.

Adenocarcinoma↗