Interaction Between Normal and Transformed Bovine Fibroblasts in Culture

1968 ◽  
Vol 3 (4) ◽  
pp. 603-613
Author(s):  
J. PONTÉN ◽  
ELIZABETH H. MACINTYRE
Keyword(s):  
Dna Synthesis ◽  
Tritiated Thymidine ◽  
Polyoma Virus ◽  
Mixed Cultures ◽  
Transformed Cells ◽  
Normal Cells ◽  
Stationary Layer

Bovine fibroblasts transformed by polyoma virus have been cocultivated with syngeneic or allogeneic normal cells. The growth of the transformed cells was inhibited by the presence of normal cells. The strongest depression was obtained when a dense stationary layer of normal fibroblasts was challenged by polyoma cells. The inhibition was least pronounced if normal and transformed cells were simultaneously seeded. Polyoma-transformed cells were identified by radioautography after incorporation of tritiated thymidine. After 96 h of contact with stationary normal cells the number of grains per nucleus and the number of labelled nuclei had not changed indicating strong depression of DNA synthesis. A deficient attachment or premature detachment of polyoma cells could not account for the observed failure of growth in the normal/polyoma mixed cultures.

Effect of cytochalasin B on somatic cell hybrids between normal and transformed cells

1985 ◽  
Vol 78 (1) ◽  
pp. 87-96
Author(s):  
I. Hickey ◽  
C. McConville ◽  
M. McMenamin ◽  
R. Neill
Keyword(s):  
Dna Synthesis ◽  
Somatic Cell ◽  
Cytochalasin B ◽  
Nuclear Division ◽  
Transformed Cells ◽  
Somatic Cell Hybrids ◽  
Recessive Character ◽  
Normal Cells ◽  
Cell Hybrids ◽  
Transformed Cell Lines

Cytochalasin B (CB) prevents cytokinesis in animal cells. In normal cells nuclear division and DNA synthesis are also blocked and the cells, held in the G1 phase of the cell cycle, remain either mononucleate or binucleate. In transformed cell lines DNA synthesis and nuclear division continue and the cells become multinucleate. We have examined the response to CB in two sets of somatic cell hybrids made between cells that display multinucleation after CB treatment and cells that do not. In a cross between transformed mouse LMTK cells and normal rat embryo lung cells, very little multinucleation was observed after treatment with CB for 7 days. The ability of the LMTK cells to form clones in soft agar was also significantly reduced in these hybrids. Segregant sub-clones that re-expressed both of these transformation phenotypes were isolated. These had reduced chromosome numbers. A second cross was made between two variants of the BHK cell line, one of which displayed a high level of multinucleation in CB while the other did not. Again the hybrids showed a response similar to that of the non-multinucleating parent. From the results obtained with these two hybrids we conclude that the multinucleation induced in transformed cells by CB behaves as a recessive character in crosses with normal cells.

scholarly journals Increased phosphorylation of ribosomal protein S6 in hamster fibroblasts transformed by polyoma virus and simian virus 40

1981 ◽  
Vol 198 (1) ◽  
pp. 235-237 ◽  
Author(s):  
Iain M. Kennedy ◽  
David P. Leader
Keyword(s):  
Ribosomal Protein ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Polyoma Virus ◽  
Transformed Cells ◽  
Normal Cells ◽  
Ribosomal Protein S6

The extent of phosphorylation of ribosomal protein S6 was compared in normal hamster fibroblasts and in fibroblasts transformed by polyoma virus or simian virus 40. In both strains of transformed cells the protein was more highly phosphorylated than in the normal cells.

Cellular targets for SV40 Large T-antigen

1985 ◽  
Vol 226 (1242) ◽  
pp. 25-42 ◽  
Keyword(s):  
Dna Synthesis ◽  
Dna Sequences ◽  
P53 Protein ◽  
T Antigen ◽  
Transformed Cells ◽  
Large T Antigen ◽  
Viral Dna ◽  
Normal Cells ◽  
Wide Range ◽  
Cellular Dna

SV40 virus infection is able to induce tumours in newborn hamsters and to transform a wide range of eukaryotic cells in in vitro culture. This is achieved by integration of the viral DNA into the host cell DNA and expression of the virus-encoded Large T-antigen. The expression of Large T, a 708 amino acid phosphoprotein, is required both to induce and maintain the transformed state. The Large T protein initiates viral DNA synthesis and regulates viral transcription, apparently by binding in a specific manner to viral DNA sequences at and near the viral origin of replication. SV40 Large T also affects cellular DNA synthesis and transcription and this may account for its oncogenic activity. A novel immunochemical procedure has permitted the isolation of cellular DNA sequences occupied by SV40 Large T in the chromatin of SV40 transformed cells. Some of the cellular sequences contain high affinity binding sites for SV40 Large T, and hybridize to messenger RNAs expressed in SV40 transformed but not in normal cells. A second type of cellular target for Large T is the cell coded p53 protein that it binds to and stabilizes. A range of monoclonal antibodies to p53 has been isolated and characterized. They demonstrate that p53 is in the cytoplasm of normal cells but is located in the nucleus of transformed cells. One of the antibodies recognizes an epitope on p53 that is stabilized or induced by binding to Large T. Further studies on the T-p53 protein complex have been facilitated by constructing bacterial plasmids that direct the synthesis of substantial quantities of Large T-β-galactosidase and p53-β-galactosidase fusion proteins in bacteria. The results are discussed in the context of our current knowledge of oncogene action.

scholarly journals Inducers of DNA synthesis: levels higher in transformed cells than in normal cells.

1983 ◽  
Vol 96 (2) ◽  
pp. 571-576 ◽  
Author(s):  
P N Rao ◽  
K L Satya-Prakash
Keyword(s):  
Dna Synthesis ◽  
Sendai Virus ◽  
Transformed Cells ◽  
G1 Phase ◽  
Normal Cells ◽  
Diploid Fibroblasts ◽  
Human Diploid Fibroblasts ◽  
Tumor Viruses ◽  
Latex Beads ◽  
Normal Human

The objective of this study was to determine whether transformed cells have greater DNA synthesis-inducing ability (DSIA) than normal cells when fused with G1 phase cells. HeLa cells synchronized in G1 phase, prelabeled with large latex beads, were fused separately with (a) quiescent human diploid fibroblasts (HDF), (b) HDF partially synchronized in late G1, and random populations of (c) HeLa, (d) WI-38, (e) SV-40 transformed WI-38, (f) CHO, (g) chemically transformed mouse cells (AKR-MCA), and (h) T98G human glioblastoma cells (all prelabeled with small latex beads) using UV-inactivated Sendai virus. The fusion mixture was incubated with [3H] thymidine, sampled at regular intervals, and processed for radioautography. Among the heterodikaryons, the frequency of those with a labeled and an unlabeled nuclei (L/U) were scored as a function of time after fusion. The faster the induction of DNA synthesis in HeLa G1, the steeper the drop in the L/U class and hence the higher DSIA in the S phase cells. The DSIA, which is indicative of the intracellular levels of the inducers of DNA synthesis, was the highest in HeLa and virally transformed WI-38 cells and the lowest in normal human diploid fibroblasts (HDF) while those of chemically and spontaneously transformed cells are intermediate between these two extremes. Higher level of DNA synthesis inducers appears to be one of the pleotropic effects of transformation by DNA tumor viruses. These studies also revealed that initiation of DNA synthesis per se is regulated by the presence of inducers and not by inhibitors.

Transfer of Growth Inhibition Between Normal and Virus-Transformed Cells: Autoradiographic Studies Using Marked Cells

1967 ◽  
Vol 2 (3) ◽  
pp. 293-304
Author(s):  
M. G. P. STOKER
Keyword(s):  
Growth Inhibition ◽  
Mouse Embryo ◽  
Normal Mouse ◽  
Growth Regulation ◽  
Thymidine Incorporation ◽  
Mixed Cultures ◽  
Transformed Cells ◽  
Normal Cells ◽  
Embryo Cells ◽  
Marking Technique

[3H]Thymidine and [3H]hypoxanthine incorporation were investigated by autoradiography in mixed cultures of polyoma-transformed BHK21 cells and freshly isolated mouse fibroblasts, with ingested carbon or carmine granules as markers to distinguish the cells. An assessment of the marking technique showed that there was some exchange of granules in the mixed cultures which prevented certain identification of individual cells, but suitable criteria were chosen for distinguishing the cells on a statistical basis. Thymidine incorporation was inhibited in one third to two thirds of the transformed cells when they were in contact with stationary layers of normal cells, which themselves showed a low proportion with thymidine incorporation. Transformed cells in the same dish which were not touching the normal cells showed no inhibition of thymidine incorporation. This is in agreement with the earlier observation that growth of transformed BHK21 cells is inhibited by contact with stationary normal fibroblasts. Experiments were also carried out on hypoxanthine incorporation with the TG1 variant of polyoma-transformed cells. TG1 cells are deficient in inosinic pyrophosphorylase and autoradiography shows a failure to incorporate hypoxanthine. When TG1 cells were cultured in contact with normal mouse embryo cells, however, it was found that hypoxanthine was present in the TG1 cells as well as in the normal cells. Increased incorporation did not occur in TG1 cells in the same dish which were not in contact with normal cells. This confirms earlier observations and shows that certain substances can pass directly from normal to transformed cells. It suggests the possibility that molecules concerned in growth regulation might also be transferred directly between contiguous cells.

scholarly journals Role of the Three Polyoma Virus Early Proteins in Tumorigenesis

1983 ◽  
Vol 3 (8) ◽  
pp. 1451-1459 ◽  
Author(s):  
Claude Asselin ◽  
Celine Gelinas ◽  
Marcel Bastin
Keyword(s):  
Cultured Cells ◽  
Virus Genome ◽  
T Antigen ◽  
Polyoma Virus ◽  
Transformed Cells ◽  
Newborn Rats ◽  
Wild Type ◽  
Complementary Function ◽  
Small T Antigen ◽  
Middle T Antigen

A modified polyoma virus genome which can encode the middle T protein but not the large or small T proteins transforms rat cells in culture with an efficiency about 20% that of the wild-type genome. Although middle T-transformed cells grow as tumors when transplanted into nude mice or syngeneic rats, the middle T gene alone is totally inactive when used in a more stringent and rigorous assay for tumorigenicity such as the injection of DNA into newborn rats. Thus, functions other than those expressed by middle T antigen are required for the elaboration of all the properties associated with tumorigenesis. To assess whether a complementary function could be exerted by the large or the small T antigen, we constructed plasmids containing two modified early regions which independently encoded middle T and one of the two other proteins. Both recombinants were tumorigenic in newborn rats. Cell lines derived by transfer of these plasmids under no special selective conditions did not acquire the property of growth in low-serum medium but exhibited the same tumorigenic properties as wild-type polyoma DNA-transformed cells. Furthermore, a recombinant which encoded the middle and small T antigens, but not the large T antigen, was tumorigenic in newborn rats. Although the small T antigen provides a complementary function for tumorigenicity, it cannot complement the middle T antigen for an efficient induction of transformation of cultured cells. This suggests that the complementary function exerted by the small T antigen is different from that of the N-terminal fragment of the large T protein.

The effect of factors released from the tumor-transformed cells on DNA synthesis, mitosis, and cellular enlargement in 3T3 fibroblasts

1987 ◽  
Vol 132 (2) ◽  
pp. 295-302 ◽  
Author(s):  
Eva Dafgård ◽  
Wilhelm Engström ◽  
Olle Larsson ◽  
Anders Zetterberg
Keyword(s):  
Dna Synthesis ◽  
Transformed Cells ◽  
3T3 Fibroblasts

A STUDY OF BLASTOCYSTS DURING DELAY AND SUBSEQUENT IMPLANTATION IN LACTATING MICE

1968 ◽  
Vol 42 (3) ◽  
pp. 453-463 ◽  
Author(s):  
ANNE McLAREN
Keyword(s):  
Dna Synthesis ◽  
Thymidine Incorporation ◽  
Tritiated Thymidine ◽  
Uterine Horn ◽  
Uterine Epithelium ◽  
Serial Sections ◽  
Uterine Lumen ◽  
Fresh State

SUMMARY Blastocysts were studied on the 5th and 8th day of pregnancy in lactating mice, in the fresh state, flushed from the uterus, in squash preparations and in serial sections. At the earlier period some mitosis was observed. Tritiated thymidine incorporation studies gave some evidence of DNA synthesis on the 5th and 6th days of pregnancy. By the 8th day the blastocysts were longer, contained more cells, and mitosis had ceased. They were located at the anti-mesometrial end of the uterine lumen, closely apposed to the uterine epithelium, and with their long axes parallel to the long axis of the uterine horn. Implantation could be induced, either by the removal of the litter, or by the injection of an appropriate dose of oestrogen on the 5th or 7th (but not the 4th) day of pregnancy. Both treatments were followed by the appearance of W-bodies in the neighbourhood of the blastocysts, the disappearance of the shed zonae, and the appearance of Pontamine Blue reactivity, oedema of the uterine stroma and formation of the primary decidual zone, in that order.

Plasminogen activator: analysis of enzyme induction by ultraviolet irradiation mapping

1981 ◽  
Vol 1 (10) ◽  
pp. 884-890
Author(s):  
R Miskin ◽  
E Reich ◽  
K Dixon
Keyword(s):  
Plasminogen Activator ◽  
Cross Sections ◽  
Ultraviolet Irradiation ◽  
Chicken Embryo ◽  
Phorbol Esters ◽  
Transformed Cells ◽  
Normal Cells ◽  
Embryo Fibroblasts ◽  
Mapping Techniques ◽  
Chicken Embryo Fibroblasts

Ultraviolet irradiation mapping techniques have previously been used to study the organization of eucaryotic gene classes and transcription units. We used the same method to probe some regulatory phenomena observed in the induction of plasminogen activator (PA) biosynthesis: PA synthesis in chicken embryo fibroblasts is induced by tumor-promoting phorbol esters and by retinoic acid; furthermore, PA induction by phorbol esters is synergistic with transformation, being 10- to 20-fold greater in virus-transformed cells than in normal cells. We found that the ultraviolet irradiation inactivation cross sections for PA induction by phorbol esters and by retinoate differed significantly, suggesting that these agents induce PA biosynthesis by different mechanisms. On the other hand, the ultraviolet irradiation sensitivity of phorbol ester induction in normal chicken embryo fibroblasts was the same as in transformed cells, indicating that the synergism of transformation and phorbol esters is probably not due to different pathways of PA induction.

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