An Inhibitor Present in Calf Serum Which Prevents Growth of BHK21 Cells in Suspension Culture

1972 ◽  
Vol 10 (1) ◽  
pp. 137-152
Author(s):  
H. OTSUKA
Keyword(s):  
Suspension Culture ◽  
Calf Serum ◽  
Soft Agar ◽  
Transformed Cells ◽  
Ammonium Sulphate Precipitation ◽  
Serum Inhibitor ◽  
Swine Serum ◽  
Rna And Dna Synthesis ◽  
Rna And Dna ◽  
Do So

BHK21 cells do not form colonies in soft agar suspension culture in the presence of calf serum; but if swine serum is used instead they do so. In methylcellulose suspension culture supplemented with calf serum the cells synthesize very little RNA or DNA, but if swine serum is used there is a marked increase of syntheses. Calf serum was found to contain an inhibitor which prevents the induction, but not the continuation, of RNA and DNA synthesis in BHK21 cells. In contrast the growth of polyoma-virus-transformed cells is not affected by this serum inhibitor which has been partially purified from calf serum by ammonium sulphate precipitation followed by Sephadex G-200 column chromatography.

Regulation of pp60c-src synthesis by inducible RNA complementary to c-src mRNA in polyomavirus-transformed rat cells

1986 ◽  
Vol 6 (7) ◽  
pp. 2305-2316
Author(s):  
S Amini ◽  
V DeSeau ◽  
S Reddy ◽  
D Shalloway ◽  
J B Bolen
Keyword(s):  
Resistance Gene ◽  
Tumor Antigen ◽  
Kinase Activity ◽  
Src Kinase ◽  
Soft Agar ◽  
Transformed Cells ◽  
Neomycin Resistance Gene ◽  
Sense Orientation ◽  
Normal Rat ◽  
Neomycin Resistance

To determine the potential role of pp60c-src in polyomavirus-transformed cells, we constructed a recombinant plasmid with the mouse metallothionein-I promoter upstream of a src gene in an anti-sense orientation. We cotransfected this plasmid into middle tumor antigen-transformed FR3T3 cells with a plasmid containing the neomycin resistance gene, and G418 resistant colonies were selected. Analysis of these cells for pp60c-src expression revealed that 50 of the 200 cellular clones screened were found to have decreased levels of c-src expression when compared with the parental middle tumor antigen-transformed cells. Three independent clones which transcribed the expected 3.6-kilobase src complementary RNA and had levels of pp60c-src kinase activity comparable to that of normal FR3T3 cells were further analyzed. In the presence of Cd2+, these clones grew significantly slower in monolayer cultures than either the parental transformed cells (FR18-1) or FR18-1 cells transfected with the neomycin resistance gene alone. The morphology of these clones in the presence of Cd2+ was distinct from that of either the parental FR18-1 cells or normal FR3T3 cells. The clones expressing the complementary src RNA were found to form fewer colonies in soft agar, form fewer foci on monolayers of normal rat cells, and form tumors more slowly following injection into syngenic rats when compared with parental FR18-1 cells. The results of these studies suggest that the level of pp60c-src kinase activity affects the growth characteristics and transformation properties of polyoma virus-transformed rat cells.

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