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.

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.5.4.642 ◽

1985 ◽

Vol 5 (4) ◽

pp. 642-648 ◽

Cited By ~ 26

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.

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Isolation of cellular genes differentially expressed in mouse NIH 3T3 cells and a simian virus 40-transformed derivative: growth-specific expression of VL30 genes.

Molecular and Cellular Biology ◽

10.1128/mcb.5.10.2590 ◽

1985 ◽

Vol 5 (10) ◽

pp. 2590-2598 ◽

Cited By ~ 21

Author(s):

K Singh ◽

S Saragosti ◽

M Botchan

Keyword(s):

Cell Lines ◽

Simian Virus 40 ◽

Mouse Cell ◽

Simian Virus ◽

Differentially Expressed ◽

Transformed Cells ◽

3T3 Cells ◽

Transformed Cell ◽

Nih 3T3 ◽

Nih 3T3 Cells

We constructed and screened a cDNA library made from simian virus 40 (SV40)-transformed NIH 3T3 cells, and we isolated cDNAs representing genes that are differentially expressed between the parental cell and its SV40-transformed derivative. We found only a small number of cDNAs representing such genes. Two isolated cDNA clones represented RNAs expressed at elevated levels in the transformed cell line in a manner relatively independent of growth conditions. The expression of two other cDNAs was growth specific because transformed cells and nonconfluent parental cells contained higher levels of the homologous RNAs than did confluent, contact-inhibited parental cells. Another cDNA was well expressed in confluent parental and confluent transformed cells, but not in nonconfluent cells. The expression of some of these cDNAs varied strikingly in different mouse cell lines. Thus the genotype or histories of different cell lines can also affect the expression of certain genes. Interestingly, the only cDNA isolated that was expressed exclusively in the transformed cell was from an SV40 message. We focused on a growth-specific cDNA which we show is derived from a mouse endogenous retrovirus-like family called VL30. We sequenced the 3' long terminal repeat (LTR) of this transcriptionally active VL30 gene. This LTR has good homology with other VL30 LTR sequences, but differences occur, particularly upstream of the VL30 promoter. We found that VL30 gene expression varied in different mouse cell lines such that C3H cell lines had very low levels of VL30 transcripts relative to NIH 3T3 cell lines. However, Southern analysis showed that both cell lines had about the same number of VL30 genes homologous to our probe and that the position of the majority of these genes was conserved. We discuss possible explanations for this difference in VL30 expression.

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Establishment of two rabbit mammary epithelial cell lines with distinct oncogenic potential and differentiated phenotype after microinjection of transforming genes.

Molecular and Cellular Biology ◽

10.1128/mcb.6.6.1974 ◽

1986 ◽

Vol 6 (6) ◽

pp. 1974-1982 ◽

Cited By ~ 21

Author(s):

I Garcia ◽

B Sordat ◽

E Rauccio-Farinon ◽

M Dunand ◽

J P Kraehenbuhl ◽

...

Keyword(s):

Epithelial Cell ◽

Cell Line ◽

Cell Lines ◽

Doubling Time ◽

Simian Virus 40 ◽

Simian Virus ◽

Oncogenic Potential ◽

Transformed Cell Line ◽

Epithelial Cell Lines ◽

Transforming Genes

The goal of this work was to establish an assay for transformation of epithelial cells. Two epithelial cell lines were obtained after microinjecting transforming genes into primary rabbit mammary secretory cells. The cell lines were analyzed for their oncogenic potential and for the maintenance of a differentiated phenotype. A fully transformed cell line, which retained epithelial cell organization, was obtained by coinjecting simian virus 40 DNA and the activated human c-Ha-ras gene. The proliferation rate of these cells was high, with a doubling time of 16 h. Their growth was anchorage independent, and they had lost contact inhibition. The cells were tumorigenic in nude mice, but had no metastatic potential. Both microinjected DNAs were efficiently transcribed and translated, in contrast to the casein genes, which were expressed in primary cells but not in the transformed cell line. An immortalized cell line established after injection with simian virus 40 DNA alone was characterized by a moderate rate of proliferation with a doubling time of approximately 30 h. The growth of these cells was contact inhibited and anchorage dependent. The cells were not tumorigenic in nude mice. The viral DNA was expressed during early passages, as shown by the presence of the large T antigen in cell nuclei, but not at later passages. A high number of lactogenic hormone receptors were found associated with the cell surface. Despite the presence of these receptors, no induction of genes coding for milk proteins was observed after addition of prolactin. These data demonstrate that this assay system can be used to assess the immortalizing and transforming potential of candidate oncogenes in epithelial cells.

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

Molecular and Cellular Biology ◽

10.1128/mcb.8.6.2668 ◽

1988 ◽

Vol 8 (6) ◽

pp. 2668-2673 ◽

Cited By ~ 54

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.

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Expression of the mouse p53 cellular tumor antigen in monkey cells

Molecular and Cellular Biology ◽

10.1128/mcb.4.10.2180-2186.1984 ◽

1984 ◽

Vol 4 (10) ◽

pp. 2180-2186

Author(s):

O Pinhasi ◽

M Oren

Keyword(s):

Tumor Antigen ◽

Recombinant Dna ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Transformed Cells ◽

Reading Frame ◽

Recombinant Plasmids ◽

Cos Cells ◽

In Cells

DNA specific for the murine p53 cellular tumor antigen was linked to the early simian virus 40 promoter and introduced into monkey COS cells either by transfection with recombinant plasmids or by infection with virus. Recipient cells made substantial amounts of a protein apparently identical to mouse p53. Severalfold-larger quantities were detected when cells were transfected with an intron-containing p53-specific segment, as compared with transfection with intronless cDNA. The p53 encoded by the recombinant DNA was capable of complexing with the simian virus 40 T antigen. Transfected p53 was also probably associated with a cellular 68-kilodalton protein, which may be related to a protein coprecipitating with p53 in some transformed cells. These findings confirm the predicted reading frame and protein boundaries and demonstrate that apparently functional p53 can be produced in cells via experimentally introduced recombinant DNA.

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Expression of the mouse p53 cellular tumor antigen in monkey cells.

Molecular and Cellular Biology ◽

10.1128/mcb.4.10.2180 ◽

1984 ◽

Vol 4 (10) ◽

pp. 2180-2186 ◽

Cited By ~ 14

Author(s):

O Pinhasi ◽

M Oren

Keyword(s):

Tumor Antigen ◽

Recombinant Dna ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Transformed Cells ◽

Reading Frame ◽

Recombinant Plasmids ◽

Cos Cells ◽

In Cells

DNA specific for the murine p53 cellular tumor antigen was linked to the early simian virus 40 promoter and introduced into monkey COS cells either by transfection with recombinant plasmids or by infection with virus. Recipient cells made substantial amounts of a protein apparently identical to mouse p53. Severalfold-larger quantities were detected when cells were transfected with an intron-containing p53-specific segment, as compared with transfection with intronless cDNA. The p53 encoded by the recombinant DNA was capable of complexing with the simian virus 40 T antigen. Transfected p53 was also probably associated with a cellular 68-kilodalton protein, which may be related to a protein coprecipitating with p53 in some transformed cells. These findings confirm the predicted reading frame and protein boundaries and demonstrate that apparently functional p53 can be produced in cells via experimentally introduced recombinant DNA.

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ESTABLISHMENT AND CHARACTERIZATION OF ETHIDIUM BROMIDE RESISTANCE IN SIMIAN VIRUS 40-TRANSFORMED HAMSTER CELLS

The Journal of Cell Biology ◽

10.1083/jcb.58.1.11 ◽

1973 ◽

Vol 58 (1) ◽

pp. 11-26 ◽

Cited By ~ 20

Author(s):

Wolfgang Klietmann ◽

Nobuhiro Sato ◽

Margit M. K. Nass

Keyword(s):

Mitochondrial Dna ◽

Cell Line ◽

Cell Lines ◽

Ethidium Bromide ◽

Simian Virus 40 ◽

Simian Virus ◽

Ultrastructural Changes ◽

Parental Cell Line ◽

Mammalian Cell Lines

This study describes the isolation and subsequent characterization of four mammalian cell lines resistant to ethidium bromide (EB). Treatment of the simian virus 40- (SV40) transformed hamster cell line F5-1 first led to the establishment of the F2 cell line, which is resistant to 2 µg EB/ml. At this concentration cytochromes c and b are present in almost normal or only slightly diminished amounts, whereas cytochromes a + a3 show an obvious decrease. The mitochondria of the F2 cell show a normal ultrastructure, not distinct from the parental cell line F5-1, and contain closed circular DNA. The sensitive parental F5-1 cells, however, when exposed to the same dye concentration exhibit the typical EB-induced ultrastructural changes in the mitochondria, and no more component I mitochondrial DNA can be demonstrated. 1 yr after establishment we derived from the F2 cell three more cell lines, resistant against 4, 8, and 16 µg of EB/ml. These cell lines, termed F4, F8, and F16, respectively, also revealed relatively intact-appearing mitochondria, although distinguishable from F5-1 and F2 mitochondria by a more condensed or unorthodox cristae conformation. F4, F8, and F16 cell lines contained closed circular mitochondrial DNA in the same position as that of the parental F5-1 cells, when analyzed in an isopycnic CsCl-EB gradient. A small shoulder at the lower density side of the DNA I peaks was observed. The newly acquired drug resistance of the F cells is hereditarily transmitted to the progeny cells and retained even after a period of growth in EB-free medium.

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Simian virus 40 large tumor antigen is unable to transform mouse embryonic fibroblasts lacking type 1 insulin-like growth factor receptor

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.90.23.11217 ◽

1993 ◽

Vol 90 (23) ◽

pp. 11217-11221 ◽

Cited By ~ 365

Author(s):

C Sell ◽

M Rubini ◽

R Rubin ◽

J P Liu ◽

A Efstratiadis ◽

...

Keyword(s):

Growth Factor ◽

Tumor Antigen ◽

Simian Virus 40 ◽

Soft Agar ◽

Simian Virus ◽

Wild Type ◽

Large Tumor ◽

Large Tumor Antigen ◽

Igf I

Fibroblast cell lines were established from mouse embryos homozygous for a targeted disruption of the Igf1r gene, encoding the type 1 receptor for insulin-like growth factor I (IGF-I) and from their wild-type littermates. The cells from the wild-type embryos (W cells) grow in serum-free medium supplemented with platelet-derived growth factor, epidermal growth factor, and IGF-I, whereas the cells from Igf1r(-/-) embryos (R- cells) do not, although they grow at a reduced rate in 10% fetal calf serum. The simian virus 40 (SV40) large T antigen, expressed from a transfected plasmid, can transform W cells, which form foci in monolayer cultures and colonies in soft agar (anchorage-independent growth). In contrast, the SV40 large tumor antigen, although normally expressed from the transfected template, is unable to transform R- cells, which remain contact-inhibited and fail to grow in soft agar. The transformed phenotype is restored if the R- cells carrying the SV40 large tumor antigen are stably transfected with a plasmid expressing the human IGF-I receptor. These results demonstrate that signaling via the IGF-I receptor is an indispensable component of the SV40 transformation pathway. This conclusion is further supported from the results of antisense RNA experiments with tumor cell lines showing that interference with the function of the IGF-I receptor has a profound effect on anchorage-independent growth, even under conditions that only modestly affect growth in monolayers.

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