scholarly journals L-myc cooperates with ras to transform primary rat embryo fibroblasts.

1988 ◽  
Vol 8 (6) ◽  
pp. 2668-2673 ◽  
Author(s):  
M J Birrer ◽  
S Segal ◽  
J S DeGreve ◽  
F Kaye ◽  
E A Sausville ◽  
...  
Keyword(s):  
Cell Lines ◽  
Simian Virus 40 ◽  
Soft Agar ◽  
Simian Virus ◽  
Rat Embryo ◽  
Ras Gene ◽  
Reading Frame ◽  
Continuous Cell Lines ◽  
Embryo Fibroblasts ◽  
Soft Agar Cloning

Recent molecular analysis has revealed that L-myc has several domains of extremely conserved amino acid sequence homology with c-myc and N-myc, suggesting similarity of function. We tested the biologic activity of L-myc by using an expression vector containing a cDNA clone coding for the major open reading frame in the 3.9-kilobase mRNA of L-myc under the control of a strong promoter (Moloney long terminal repeat) and found that L-myc complemented an activated ras gene in transforming primary rat embryo fibroblasts. However, the efficiency of transformation was 1 to 10% of that seen with the c-myc and simian virus 40 (SV40) controls. The L-myc/ras transformants initially grew more slowly than c-myc or SV40 transformants, but once established as continuous cell lines, they were indistinguishable from cell lines derived from c-myc/ras or SV40/ras transfectants as determined by morphology, soft-agar cloning, and tumorigenicity in nude mice.

L-myc cooperates with ras to transform primary rat embryo fibroblasts

1988 ◽  
Vol 8 (6) ◽  
pp. 2668-2673
Author(s):  
M J Birrer ◽  
S Segal ◽  
J S DeGreve ◽  
F Kaye ◽  
E A Sausville ◽  
...  
Keyword(s):  
Cell Lines ◽  
Simian Virus 40 ◽  
Soft Agar ◽  
Simian Virus ◽  
Rat Embryo ◽  
Ras Gene ◽  
Reading Frame ◽  
Continuous Cell Lines ◽  
Embryo Fibroblasts ◽  
Soft Agar Cloning

Recent molecular analysis has revealed that L-myc has several domains of extremely conserved amino acid sequence homology with c-myc and N-myc, suggesting similarity of function. We tested the biologic activity of L-myc by using an expression vector containing a cDNA clone coding for the major open reading frame in the 3.9-kilobase mRNA of L-myc under the control of a strong promoter (Moloney long terminal repeat) and found that L-myc complemented an activated ras gene in transforming primary rat embryo fibroblasts. However, the efficiency of transformation was 1 to 10% of that seen with the c-myc and simian virus 40 (SV40) controls. The L-myc/ras transformants initially grew more slowly than c-myc or SV40 transformants, but once established as continuous cell lines, they were indistinguishable from cell lines derived from c-myc/ras or SV40/ras transfectants as determined by morphology, soft-agar cloning, and tumorigenicity in nude mice.

scholarly journals Rat embryo fibroblasts immortalized with simian virus 40 large T antigen undergo senescence upon its inactivation.

1996 ◽  
Vol 16 (9) ◽  
pp. 5127-5138 ◽  
Author(s):  
E S Gonos ◽  
J S Burns ◽  
G R Mazars ◽  
A Kobrna ◽  
T E Riley ◽  
...  
Keyword(s):  
Cell Lines ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Growth Arrest ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Rat Embryo ◽  
G2 Block ◽  
Embryo Fibroblasts

Introduction of simian virus 40 T antigen into rodent fibroblasts gives rise to cells that can proliferate indefinitely but are dependent upon it for maintenance of their growth once the normal mitotic life span has elapsed. Inactivation of T antigen in these immortalized cells causes rapid and irreversible cessation of growth. To determine whether this growth arrest is associated with entry into senescence, we have undertaken a genetic and biological analysis of conditionally immortal (tsa) cell lines derived by immortalizing rat embryo fibroblasts with the thermolabile tsA58 T antigen. This analysis has identified the following parallels between the tsa cells after inactivation of T antigen and senescent rat embryo fibroblasts: (i) growth arrest is irreversible; (ii) it occurs in G1 as well as G2; (iii) the G1 block can be partially overcome by stimulation with 20% fetal calf serum, but the G2 block cannot be overcome; (iv) 20% fetal calf serum induces c-fos, but c-myc is unaltered; and (v) fibronectin and p21(Waf1/Cip1/Sdi1) are upregulated upon growth arrest. These results suggest that T-antigen-immortalized fibroblasts are committed to undergo senescence but are prevented from undergoing this process by T antigen. Inactivation of T antigen removes this block and results in senescence of the cells. Thus, these cell lines may represent a powerful system for study of the molecular basis of entry into senescence.

scholarly journals Analysis of a transgenic mouse containing simian virus 40 and v-myc sequences.

1985 ◽  
Vol 5 (4) ◽  
pp. 642-648 ◽  
Author(s):  
J A Small ◽  
D G Blair ◽  
S D Showalter ◽  
G A Scangos
Keyword(s):  
Cell Lines ◽  
Choroid Plexus Papilloma ◽  
Simian Virus 40 ◽  
Soft Agar ◽  
Simian Virus ◽  
T Antigen ◽  
Primary Cell ◽  
Tissue Samples ◽  
Primary Cell Lines

Two plasmids, one containing the simian virus 40 (SV40) genome and the mouse metallothionein I gene and one containing the v-myc gene of avian myelocytomatosis virus MC29, were coinjected into mouse embryos. Of the 13 surviving mice, one, designated M13, contained both myc and SV40 sequences. This mouse developed a cranial bulge identified as a choroid plexus papilloma at 13 weeks and was subsequently sacrificed; tissue samples were taken for further analysis. Primary cell lines derived from these tissues contained both myc and SV40 DNA. No v-myc mRNA could be detected, although SV40 mRNA was present in all of the cell lines tested. T antigen also was expressed in all of the cell lines analyzed. These data suggest that SV40 expression was involved in the abnormalities of mouse M13 and was responsible for the transformed phenotype of the primary cell lines. Primary cell lines from this mouse were atypical in that the population rapidly became progressively more transformed with time in culture based on the following criteria: morphology, growth rate, and the ability to grow in soft agar and in serum-free medium. The data also suggest that factors present in the mouse regulated the ability of SV40 to oncogenically transform most cells and that in vitro culture of cells allowed them to escape those factors.

scholarly journals Differential ability of a T-antigen transport-defective mutant of simian virus 40 to transform primary and established rodent cells.

1985 ◽  
Vol 5 (5) ◽  
pp. 1043-1050 ◽  
Author(s):  
R E Lanford ◽  
C Wong ◽  
J S Butel
Keyword(s):  
Cell Lines ◽  
Mouse Embryo ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Wild Type ◽  
Mouse Embryo Fibroblasts ◽  
Established Cell Lines ◽  
Established Cell ◽  
Embryo Fibroblasts

The transforming potential and oncogenicity of a simian virus 40 (SV40) mutant affecting T-antigen (T-ag), SV40(cT)-3, was examined in an effort to dissect T-ag functions in transformation. SV40(cT)-3 has a point mutation at nucleotide 4434 that abolishes the transport of T-ag to the nucleus but does not affect its association with the cell surface. Transfection-transformation assays were performed with primary cells and established cell lines of mouse and rat origin. The efficiency of transformation for established cell lines by SV40(cT)-3 was comparable to that of wild-type SV40, indicating that transformation of established cell lines can occur in the absence of detectable amounts of nuclear T-ag. Transformation of primary mouse embryo fibroblasts by SV40(cT)-3 was markedly influenced by culture conditions; the relative transforming frequency was dramatically reduced in assays involving focus formation in low serum concentrations or anchorage-independent growth. Immunofluorescence tests revealed that the transformed mouse embryo fibroblasts partially transport the mutant cT-ag to the cell nucleus. Transformed cell lines induced by SV40(cT)-3 did not differ in growth properties from wild-type transformants. SV40(cT)-3 was completely defective for the transformation of primary baby rat kidney cells, a primary cell type unable to transport the mutant T-ag to the nucleus. The intracellular localization of cellular protein p53 was found to mimic T-ag distribution in all the transformants analyzed. The mutant virus was weakly oncogenic in vivo: the induction of tumors in newborn hamsters by SV40(cT)-3 was reduced in incidence and delayed in appearance in comparison to wild-type SV40. These observations suggest that cellular transformation is regulated by both nuclear and surface-associated forms of SV40 T-ag.

Revertants of v-fos-transformed rat fibroblasts: suppression of transformation is dominant

1990 ◽  
Vol 10 (11) ◽  
pp. 5626-5633
Author(s):  
R Wisdom ◽  
I M Verma
Keyword(s):  
Divalent Cations ◽  
Simian Virus 40 ◽  
Soft Agar ◽  
Simian Virus ◽  
Ras Gene ◽  
Recessive Mutations ◽  
Activating Mutation ◽  
Murine Sarcoma Virus ◽  
Sarcoma Virus ◽  
Rat Fibroblasts

Phenotypic revertants of Finkel-Biskis-Riley (FBR)-murine sarcoma virus-transformed rat fibroblasts were isolated on the basis of their adherence to plastic tissue culture dishes in the absence of divalent cations. Some revertants had sustained deletions or inactivating mutations of the v-fos gene. However, two revertants expressed a functional v-fos gene at levels equal to that in the transformed parental cells, and therefore phenotypic reversion was due to mutations in nonviral genes. These revertants were considered nontransformed according to four criteria: (i) they were flat and had a nontransformed morphology, (ii) they were contact inhibited when grown to confluence, (iii) they did not display anchorage-independent growth in soft agar, and (iv) they did not form tumors in nude mice. Somatic-cell hybrids between the revertants and the transformed parental cells were nontransformed, suggesting that the revertants had sustained an activating mutation of a gene capable of suppressing transformation. The expression of c-jun, junB, and junD was not altered in the revertants, and they could not be transformed by transfection with a c-jun expression vector. The revertants were resistant to transformation by an activated c-Ha-ras gene but were susceptible to transformation by simian virus 40. Our results demonstrate the existence of a class of revertants that harbor genes capable of suppressing transformation by v-fos and some other oncogenes. This contrasts with previously described revertants of transformation by v-fos that contain recessive mutations.

scholarly journals Neoplastic transformation of rat 3Y1 cells by a transcriptionally activated human c-myc gene and stabilization of p53 cellular tumor antigen in the transformed cells.

1986 ◽  
Vol 6 (12) ◽  
pp. 4379-4386 ◽  
Author(s):  
K Shiroki ◽  
K Segawa ◽  
Y Koita ◽  
M Shibuya
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Tumor Antigen ◽  
Simian Virus 40 ◽  
Soft Agar ◽  
Simian Virus ◽  
Transformed Cells ◽  
Agar Culture ◽  
Myc Gene ◽  
Tumorigenic Cell Line

Transformed foci were obtained in rat 3Y1 fibroblasts cotransfected with pRmyc 27 (transcriptionally activated c-myc) and pSV2neo DNA. RmycY cell lines (1 to 7) were established from these foci. RmycY cells were small and round and contained enlarged nucleoli in the nucleus. The myc gene was expressed in these cell lines at a much higher level than in 3Y1 cells and at a level similar to that in HL-60 cells. These cell lines formed colonies in soft-agar culture and tumors in syngeneic rats transplanted with RmycY cells. Expression of the gene and colony formation in soft-agar culture were analyzed in subclones from RmycY cell line 1. A correlation between myc gene expression and the ability to form colonies in soft-agar culture was observed in these cells. Antibody against p53 cellular tumor antigen was detected in some sera from tumor-bearing rats. p53 cellular tumor antigen stabilized and accumulated in RmycY cells to the same extent as in simian virus 40-transformed cells. The results suggest that elevated c-myc expression and an increased amount of p53 cause 3Y1 cells to become a more tumorigenic cell line.

scholarly journals Heterologous expression and characterization of the human R-ras gene product.

1987 ◽  
Vol 7 (8) ◽  
pp. 2845-2856 ◽  
Author(s):  
D G Lowe ◽  
D V Goeddel
Keyword(s):  
Electrophoretic Mobility ◽  
Simian Virus 40 ◽  
Soft Agar ◽  
Simian Virus ◽  
Biochemical Properties ◽  
Ras Gene ◽  
Focus Formation ◽  
Dependent Change ◽  
Ras P21 ◽  
Trp Promoter

We directly expressed human R-ras 23,000-dalton protein (p23) cDNA in Escherichia coli under the control of the trp promoter. GTP-dependent phosphorylation of a p23 threonine 85 substitution mutant was observed. This result is in direct analogy to the autokinase activity of H-ras and K-ras threonine 59 substitution mutants. Normal p23 protein was detected in the human fibrosarcoma cell line HT1080 by immunoprecipitation with rabbit antibodies raised against an E. coli-expressed R-ras fusion protein. The R-ras p23 protein was found to be 3H labeled in the presence of [9,10(n)-3H]palmitic acid and is associated with the P100 membrane fraction of HT1080 cells. These data suggest that human R-ras p23 has biochemical properties very similar to those of the p21 products of the H-, K-, and N-ras proto-oncogenes. We constructed an R-ras minigene and engineered the expression of normal and mutant alleles from the simian virus 40 early region promoter. Normal and mutant R-ras gene products were authenticated by transient expression in COS-7 cells and immunoprecipitation. The valine 38-substituted R-ras p23 displayed reduced electrophoretic mobility. R-ras p21-like proteins, made by eliminating the first 26 R-ras codons, displayed evident mobility differences between the pro form and mature form, along with a valine 12 substitution-dependent change in electrophoretic mobility. Rat-1 fibroblasts were transfected with normal and mutant R-ras alleles and normal and activated H-ras alleles. Unlike the human T24 bladder oncogene-encoded p21, mutant R-ras alleles do not cause monolayer focus formation or growth in soft agar of rat fibroblasts.

scholarly journals Activated Ha-ras can cooperate with defective simian virus 40 in the transformation of nonestablished rat embryo fibroblasts.

1987 ◽  
Vol 61 (8) ◽  
pp. 2648-2654 ◽  
Author(s):  
D Michalovitz ◽  
L Fischer-Fantuzzi ◽  
C Vesco ◽  
J M Pipas ◽  
M Oren
Keyword(s):  
Simian Virus 40 ◽  
Simian Virus ◽  
Rat Embryo ◽  
Embryo Fibroblasts

scholarly journals Resistance to oncogenic transformation in revertant R1 of human ras-transformed NIH 3T3 cells.

1989 ◽  
Vol 9 (5) ◽  
pp. 2258-2263 ◽  
Author(s):  
N Kuzumaki ◽  
Y Ogiso ◽  
A Oda ◽  
H Fujita ◽  
H Suzuki ◽  
...  
Keyword(s):  
Simian Virus 40 ◽  
Soft Agar ◽  
Simian Virus ◽  
T Antigen ◽  
Oncogenic Transformation ◽  
3T3 Cells ◽  
Cellular Mechanisms ◽  
Ras Gene ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

A flat revertant, R1, was isolated from human activated c-Ha-ras-1 (hu-ac-Ha-ras) gene-transformed NIH 3T3 cells (EJ-NIH 3T3) treated with mutagens. R1 contained unchanged transfected hu-ac-Ha-ras DNA and expressed high levels of hu-ac-Ha-ras-specific mRNA and p21 protein. Transfection experiments revealed that NIH 3T3 cells could be transformed by DNA from R1 cells but R1 cells could not be retransformed by Kirsten sarcoma virus, DNA from EJ-NIH 3T3 cells, hu-ac-Ha-ras, v-src, v-mos, simian virus 40 large T antigen, or polyomavirus middle T antigen. Somatic cell hybridization studies showed that R1 was not retransformed by fusion with NIH 3T3 cells and suppressed anchorage independence of EJ-NIH 3T3 and hu-ac-Ha-ras gene-transformed rat W31 cells in soft agar. These results suggest that the reversion and resistance to several oncogenes in R1 is due not to cellular defects in the production of the transformed phenotype but rather to enhancement of cellular mechanisms that suppress oncogenic transformation.

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