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Biomedical subjects

F Birg

Publications and source records attributed to F Birg.

70 records · Page 4Linked to original sources
PubMed1984

Cell lines derived from tumors induced in syngeneic rats by FR 3T3 SV40 transformants no longer synthesize the early viral proteins.

Several SV40-transformed FR 3T3 rat cell lines formed tumors upon inoculation to syngeneic immunocompetent Fisher rats. These tumors, which appeared only after a long latency period, showed a fast rate of growth. Tumor-derived (TD) cell lines were established in culture from several tumors induced by independent transformants, and their properties were analyzed. Though TD cells were highly tumorigenic, their level of transformation in culture was similar to that of the original transformants. They did not synthesize detectable amounts of the two early viral gene products, the large-T and small-T polypeptides. However, the transformation-associated cellular p53 protein was detected in all of them by [35S]methionine labeling and immune precipitation with monoclonal antibodies directed against the mouse p53. Growth in the animal apparently counterselected the cells expressing the early viral proteins, and hence, possibly, the tumor-specific transplantation antigen. This selection was mediated at least in part by the T-cell immune response, as the tumors induced by the same transformants in nu/nu mice still expressed the nuclear T-antigen. Absence of expression of the early viral region was frequently correlated with the loss of the integrated SV40 DNA. Some tumors, however, still contained early viral DNA sequences, which were, in even fewer cases, transcribed into RNA. These results altogether suggest that tumor formation by the FR 3T3-SV40 transformed cells in immunocompetent rats requires two events, the selection for the acquisition of a high tumorigenic potential, and against the expression of the early viral genes. Only the first of these two events was observed upon tumor formation in nude mice.

Animals↗
PubMed1983

Simian virus 40 T antigen is detected only in cells in the G2 phase of the cell cycle in one group of rat transformants.

The relationship between the cell cycle and the presence of the nuclear T antigen in simian virus 40-transformed rat cells independently derived from FR 3T3 cells has been investigated using a fluorescence-activated cell sorter and a combination of stainings for cellular DNA (4',6-diamidine-2-phenylindole dihydrochloride, DAPI) and for the viral T antigen (fluorescein-labeled IgG). Such an analysis revealed that in three out of six lines, T antigen could be detected only in the G2 phase of the cell cycle. In the other three lines, the accumulation of T antigen appeared to be independent of the position of the cell in the cycle. The emergence of either phenotype was strictly correlated with the physiological state of the cell (growing versus resting, respectively) at the time the transformants were established.

Animals↗
PubMed1983

Stabilization of the large T protein in temperature-independent (type A) FR 3T3 rat cells transformed with the simian virus 40 tsA30 mutant.

The stabilities of in vivo [35S]methionine-labeled large T and small t proteins, synthesized in temperature-sensitive (type N) and temperature-insensitive (type A) FR 3T3 rat cells transformed by an early temperature-sensitive mutant of simian virus 40 (SV40), tsA30, were analyzed at the permissive and restrictive temperatures. The two polypeptides, detected in greatly reduced amounts in cells of the N type at the restrictive temperature, were also unstable at the permissive temperature. However, both were made in similar amounts and were apparently stable in cells of the A type, irrespective of the temperature. The structures of the viral RNAs present at the permissive temperature were analyzed for transformants representative of each type, and containing a single integration of viral DNA. The two cell lines synthesized transcripts identical to the large T and small t mRNAs identified in SV40-infected monkey cells. Similar amounts of viral RNA were found in A and N transformants in active growth at the permissive and restrictive temperatures, which argued against a control at a transcriptional level. Assay of a defined function of the protein, namely, the binding of nucleotide detected by affinity labeling with periodate-oxidized [alpha-32P]ATP, clearly showed that the large T proteins from both types of transformants exhibited, at least for that particular biochemical function, the same in vitro temperature sensitivity. In transformants of the A type only could a reduced binding activity be detected in extracts from cells grown at the restrictive temperature. Thus, the temperature-independent behavior of the A transformants may result from an in vivo partial stabilization of the newly synthesized large T protein, probably through interaction with a cellular component(s).

Animals↗
PubMed1980

Integration sites and sequence arrangement of SV40 DNA in a homogeneous series of transformed rat fibroblast lines.

The state and organization of viral DNA sequences present in independently isolated rat cell lines transformed with SV40 were investigated using restriction-enzyme cleavage of the cellular DNA and blot hybridization with a viral probe. The transformed lines were established under conditions as identical as possible, except for a limited number of variables (multiplicity of infection, physiological state of the cells after infection and procedures used for selecting the transformed derivatives). They were characterized after a limited number of generations in culture. Two distinct types of organization were found: covalently integrated viral genomes were present either as single inserts or as head-to-tail oligomeric structures. The latter was observed among transformants derived from cells maintained after infection under growth-inhibiting conditions (suspension in agarose medium, confluency on a solid substrate). Single inserts were observed only among cell lines isolated after an initial period of active growth. Recurrent patterns of hybridizaton were observed in independently isolated lines, indicating that the sites of the integrative recombinations were close enough, both in the viral and the cellular sequences, not to be distinguished at the level of sensitivity of the technique (more than +/- 100 bp). Among cell lines with multiple integration sites, only part of the inserts were found in several instances to be identical to inserts observed in other transformed lines.

Animals↗
PubMed1979

State and organization of polyoma virus DNA sequences in transformed rat cell lines.

Polyoma virus-transformed rat cell lines were isolated as colonies growing in agar after infection of F2408 cells with low multiplicities of wild-type virus. Viral DNA present in the transformed cells was analyzed by fractionating the cellular DNA on agarose gels before and after digestion with various restriction endonucleases, followed by detection of the DNA fragments containing viral sequences using the procedure described by Southern (E. Southern, J. Mol. Biol., 98:503--515, 1975). Five lines, independently derived, were studied in detail. All five lines, when examined after a minimum number of passages in culture, contained both free and apparently integrated viral DNA. The free polyoma DNA in three of the lines was indistinguishable, by restriction enzyme analysis, from wild-type viral DNA, whereas the two other lines also contained smaller free DNA molecules which lacked parts of the wild-type genome. The integrated DNA in the five lines studies existed as head-to-tail tandem repeats of unit-length polyoma DNA covalently attached to nonviral DNA. The same five polyoma-transformed rat lines were examined after further passage in culture. Free viral DNA was then either undetectable or greatly reduced in amounts, whereas the high-molecular-weight, integrated units persisted after passage of the cells. The subclones, derived from one of the five lines selected for detailed analysis, showed some variations in the quantity and size of the free viral DNA as well as minor alterations in the pattern of the apparently integrated sequences.

Animals↗
PubMed1977

Analysis of polyoma virus nuclear RNA by mini-blot hybridization.

The size and sequence composition of virus-specific RNA extracted from the nuclei of mouse cells late during polyoma virus productive infection were studied by blot-hybridization analysis of 32P-labeled RNA fractionated on CH3HgOH/agarose gels. Viral RNA molecules between approximately 0.4 and 4 times the length of a complete transcript of the 5.4-kilobase circular viral DNA were found. Less than 20% of such molecules were polyadenylylated. Although viral RNA of all sizes contained species that together hybridized to the entire polyoma genome, sequences complementary to the late region were more abundant than sequences complementary to the early region in transcripts less than 10-12 kilobases long.

Base Sequence↗
PubMedDate not supplied

A defective retroviral vector encoding human interferon-alpha2 can transduce human leukemic cell lines.

Using the LXSN backbone, a defective retroviral vector (LISN) was constructed that encodes the human interferon (IFN)-alpha2 (hIFN-alpha2) gene and the neomycin resistance gene; the hIFN-alpha2 gene was cloned from human placental genomic DNA. High titers of the LISN retrovirus were produced by the amphotropic packaging cell line GP+envAM12. LISN is able to infect three human hematopoietic and leukemic cell lines: K562, LAMA-84, and TF-1. G418-resistant cells were detected in a similar proportion after infection with either the LISN retroviral vector or the LnLSN retroviral vector (encoding the nlsLacZ gene instead of hIFN-alpha2), suggesting that hIFN-alpha2 does not inhibit (or only partially inhibits) the production of retroviral particles by the packaging cell line and the infection of human cells. LISN-infected cells express and secrete hIFN-alpha2 as demonstrated by Northern blot analysis of poly(A)+ RNA, detection of the intracellular protein by fluorescence-activated cell sorter analysis, and detection of secreted hIFN-alpha in cell supernatants using an enzyme-linked immunosorbent assay. Retrovirally produced hIFN-alpha2 is biologically active, as demonstrated by the partial inhibition of the growth of K562 and TF-1, the modulation of the expression of cell surface antigens, the induction of the (2'-5') oligoadenylate synthetase, and, for LAMA-84, the down-modulation of the BCR-ABL protein. We conclude that the infection of human leukemic cell lines with a retroviral vector encoding hIFN-alpha2 is feasible and induces the expected biological effects. This experimental model will be useful in investigating the possibility of transducing normal and leukemic cells and hematopoietic progenitors and in determining the consequences of the autocrine production of hIFN-alpha2 on the behavior of these cells.

Animals↗