scholarly journals Quantitative biochemical analysis of microtubule content in normal and transformed 3T3 cells.

1979 ◽  
Vol 82 (2) ◽  
pp. 572-576 ◽  
Author(s):  
J H Eichhorn ◽  
B Peterkofsky
Keyword(s):  
Cell Density ◽  
Cell Lines ◽  
Cell Morphology ◽  
Biochemical Analysis ◽  
Transformed Cells ◽  
Dibutyryl Camp ◽  
3T3 Cells ◽  
Normal Cells ◽  
Stages Of Growth ◽  
Tubulin Content

Microtubules in normal and transformed BALB 3T3 cells were preserved in a stabilizing medium and measured by a [3H]colchicine-binding tubulin assay, and compared to total cellular tubulin measured under nonstabilizing conditions. Essentially no change in tubulin or microtubule content was seen with changes in cell density or with changes in cellular morphology at various stages of growth of normal or transformed cells or induced by dibutyryl cAMP treatment of transformed cells. Of five cell lines transformed by a variety of agents, four had a significantly higher total tubulin content than untransformed 3T3 cells and all of them had an increased microtubule content. None of the transformed lines had a lower fraction of tubulin recoverable as sedimentable microtubules compared to untransformed cells, and in three of them this fraction was significantly higher. These results establish that microtubules are present in transformed cells to at least the extent (if not greater) than in normal cells but that there are variations in the total amount of tubulin and microtubules as well as the fraction of the total tubulin present as microtubules which are not strictly correlated with transformation or cell morphology.

scholarly journals Identification of a BALB/c-3T3 cell protein modulated by platelet-derived growth factor.

1983 ◽  
Vol 3 (1) ◽  
pp. 70-81 ◽  
Author(s):  
C D Scher ◽  
R L Dick ◽  
A P Whipple ◽  
K L Locatell
Keyword(s):  
Growth Factor ◽  
Dna Synthesis ◽  
Cell Lines ◽  
Actinomycin D ◽  
Platelet Derived Growth Factor ◽  
Cell Protein ◽  
Transformed Cells ◽  
Antigenic Determinants ◽  
3T3 Cells ◽  
Constitutive Synthesis

The platelet-derived growth factor (PDGF) stimulates density-arrested BALB/c-3T3 cells to synthesize a protein (pII; Mr, 35,000) that is constitutively synthesized by spontaneously transformed BALB/c-3T3 (ST2-3T3) cells which do not require PDGF for growth. Antisera against a major excreted protein family (MEP) of retrovirus-transformed cells quantitatively precipitated cellular pII. PDGF-stimulated pII has the same molecular weight, a similar charge, and similar antigenic determinants as authentic MEP isolated from ST2-3T3 or retrovirus-transformed cells. MEP represented about 2% of the nonnuclear proteins synthesized by ST2-3T3 cells and 0.3 to 0.6% of the proteins synthesized by PDGF-treated BALB/c-3T3 cells, a three- to sixfold increase over the background. In BALB/c-3T3 cells, less PDGF was required for pII (MEP) synthesis than for DNA synthesis. PDGF induced a selective increase in pII (MEP) within 40 min. Such preferential synthesis was inhibited by brief treatment with actinomycin D, suggesting a requirement for newly formed RNA. The constitutive synthesis of pII (MEP) by ST2-3T3 cells was not inhibited by actinomycin D. Five spontaneously or chemical carcinogen-transformed tumorigenic BALB/c-3T3 cell lines were studied; they neither required PDGF for growth nor responded to it. These cell lines became arrested at confluence with a G1 DNA content. Each of these independently isolated lines synthesized pII (MEP) constitutively. Thus, the synthesis of pII (MEP) may be required, but is not sufficient, for PDGF-modulated DNA synthesis.

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

1985 ◽  
Vol 5 (10) ◽  
pp. 2590-2598 ◽  
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.

Cell surface topography in adhesion and detachment

1978 ◽  
Vol 36 (2) ◽  
pp. 300-301
Author(s):  
T.D. Allen
Keyword(s):  
Cell Lines ◽  
Thin Sheet ◽  
Liver Parenchyma ◽  
Soft Agar ◽  
Transformed Cells ◽  
Normal Cells ◽  
Epithelial Cell Lines ◽  
Normal Rat ◽  
Transformed Cell Lines ◽  
Peripheral Cytoplasm

The effects of trypsin and EGTA were investigated in parallel on two epithelial cell lines: a normal rat-liver parenchyma line (Fig. 1) and a spontaneously transformed line which had been subcloned in soft agar. The basic ultrastructural differences between these two cell lines has already been reported; briefly, the transformed cells, although typically epithelial in morphology, lack the well ordered microtubular and microfilamentous organisation of the normal cells but exhibit a marked increase in the number of surface microvilli.The reaction of each cell line to trypsination was broadly similar between the normal and transformed cell lines, with the transformed cells rounding up and detaching at a slightly quicker rate than the normals. In both cases the central region of the cell became domed with the peripheral cytoplasm being withdrawn in a thin sheet, some of which remained extended in short retraction fibrils (Fig. 2). Collection of the detached cells (after fixation in suspension) by aspiration onto silver filters showed the increased density of microvilli on the transformed cells to be maintained.

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

1985 ◽  
Vol 5 (10) ◽  
pp. 2590-2598
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.

Identification of a BALB/c-3T3 cell protein modulated by platelet-derived growth factor

1983 ◽  
Vol 3 (1) ◽  
pp. 70-81
Author(s):  
C D Scher ◽  
R L Dick ◽  
A P Whipple ◽  
K L Locatell
Keyword(s):  
Growth Factor ◽  
Dna Synthesis ◽  
Cell Lines ◽  
Actinomycin D ◽  
Platelet Derived Growth Factor ◽  
Cell Protein ◽  
Transformed Cells ◽  
Antigenic Determinants ◽  
3T3 Cells ◽  
Constitutive Synthesis

The platelet-derived growth factor (PDGF) stimulates density-arrested BALB/c-3T3 cells to synthesize a protein (pII; Mr, 35,000) that is constitutively synthesized by spontaneously transformed BALB/c-3T3 (ST2-3T3) cells which do not require PDGF for growth. Antisera against a major excreted protein family (MEP) of retrovirus-transformed cells quantitatively precipitated cellular pII. PDGF-stimulated pII has the same molecular weight, a similar charge, and similar antigenic determinants as authentic MEP isolated from ST2-3T3 or retrovirus-transformed cells. MEP represented about 2% of the nonnuclear proteins synthesized by ST2-3T3 cells and 0.3 to 0.6% of the proteins synthesized by PDGF-treated BALB/c-3T3 cells, a three- to sixfold increase over the background. In BALB/c-3T3 cells, less PDGF was required for pII (MEP) synthesis than for DNA synthesis. PDGF induced a selective increase in pII (MEP) within 40 min. Such preferential synthesis was inhibited by brief treatment with actinomycin D, suggesting a requirement for newly formed RNA. The constitutive synthesis of pII (MEP) by ST2-3T3 cells was not inhibited by actinomycin D. Five spontaneously or chemical carcinogen-transformed tumorigenic BALB/c-3T3 cell lines were studied; they neither required PDGF for growth nor responded to it. These cell lines became arrested at confluence with a G1 DNA content. Each of these independently isolated lines synthesized pII (MEP) constitutively. Thus, the synthesis of pII (MEP) may be required, but is not sufficient, for PDGF-modulated DNA synthesis.

scholarly journals CONTACT-INHIBITED REVERTANT CELL LINES ISOLATED FROM SV40-TRANSFORMED CELLS

1971 ◽  
Vol 50 (3) ◽  
pp. 682-690 ◽  
Author(s):  
Lloyd A. Culp ◽  
William J. Grimes ◽  
Paul H. Black
Keyword(s):  
Sialic Acid ◽  
Cell Lines ◽  
Acid Content ◽  
Inverse Correlation ◽  
Wild Type Virus ◽  
Transformed Cells ◽  
Type Virus ◽  
Wild Type ◽  
3T3 Cells ◽  
Sialic Acid Content

Two contact-inhibited "revertant" cell lines were isolated from an SV40-transformed mouse 3T3 cell line (SV-3T3) after exposure to 5-fluoro-2'-deoxyuridine. Revertant cells resembled 3T3 cells morphologically and grew to saturation densities which were similar to those of 3T3 cells; however, revertant cells readily formed both single and multinucleated giant cells in confluent cultures. SV40 virus was rescued from revertant cells by fusion with permissive monkey cells. The rescued virus transformed 3T3 cells with the same efficiency as wild type virus, and produced transformed colonies which were phenotypically similar to those produced by wild type virus. The revertant cells also resembled normal 3T3 cells in that they contained higher quantities of sialic acid than SV-3T3 cells. An inverse correlation was found between the saturation density of cells and their sialic acid content. Collagen content, however, of revertant cells was similar to that of SV-3T3 cells. The data presented suggest that the property of contact inhibition in revertant cells is related to the sialic acid content of the plasma membrane and that changes in sialic acid content of transformed cells are not directly specified by the viral genome.

Altered regulation of c-myc expression in adenovirus-transformed cells

1986 ◽  
Vol 28 (5) ◽  
pp. 712-724
Author(s):  
Antony W. Braithwaite ◽  
Kathy E. Fry ◽  
Sue LeJeune ◽  
Hiroto Naora
Keyword(s):  
Cell Cycle ◽  
Cell Density ◽  
Cell Lines ◽  
Transformed Cells ◽  
Serum Starvation ◽  
Mrna Levels ◽  
Primary Cells ◽  
Regulation Of Expression ◽  
Primary Mouse ◽  
Transformed Cell Lines

Expression of the oncogenes c-myc, c-raski, and p53 is studied in normal primary mouse cultures and in two adenovirus-transformed mouse cell lines. In all cases oncogene expression is measured in cells arrested in G1 (or G0 for primary cells) by serum starvation and at different times after cell cycle traverse is stimulated by addition of high serum. For primary mouse cells, c-myc mRNA levels are found to increase four- to six-fold within 1 h of serum addition and then decline by 4 h to nearly the level observed in serum-starved cells. This level is maintained throughout the remainder of the cell cycle. The early induction of c-myc is dependent on serum concentration and is independent of cell density. These results confirm and extend previous observations for primary cells. By contrast, expression of c-raski does not vary at all through the cell cycle and p53 increases with time after mitogenic stimulation. In the adenovirus-transformed cell lines, the regulation of expression of c-myc with respect to the cell cycle is altered. There is an increase in c-myc in S phase cells which is dependent on cell density and the early induction in response to serum addition as seen in primary cells is absent. Expressions of c-raski and p53 are found to show similar profiles to those observed for primary cells.Key words: cell cycle, c-myc, transformed cells.

Interaction Between Normal and Transformed Bovine Fibroblasts in Culture

1967 ◽  
Vol 2 (3) ◽  
pp. 309-322
Author(s):  
ELIZABETH MACINTYRE ◽  
J. PONTÉN
Keyword(s):  
Rous Sarcoma Virus ◽  
Lung Fibroblasts ◽  
Transformed Cells ◽  
Embryonic Lung ◽  
Normal Cells ◽  
Stages Of Growth ◽  
Rous Sarcoma ◽  
No Inhibition ◽  
Sarcoma Virus

Base layers of untransformed normal embryonic lung fibroblasts in different stages of growth were challenged with RSV (Rous sarcoma virus)-transformed bovine fibroblasts. The effect of this admixture on the growth of the RSV-transformed cells was studied. In all cases, the transformed cells proliferated freely, and it is concluded that the normal cells exerted no inhibition on the transformed cells.

scholarly journals Patterns of plasminogen activator production in cultured normal embryonic cells.

1977 ◽  
Vol 75 (1) ◽  
pp. 31-42 ◽  
Author(s):  
S T Rohrlich ◽  
D B Rifkin
Keyword(s):  
Plasminogen Activator ◽  
Negative Control ◽  
Transformed Cells ◽  
Embryonic Cells ◽  
3T3 Cells ◽  
Normal Cells ◽  
Serum Supplementation ◽  
Low Passage ◽  
Passage Cells ◽  
Acid Labile

Cultured normal low-passage embryo fibroblasts, from a number of species, and two untransformed clones of a Balb/3T3 line elaborate increasing amounts of plasminogen activator (PA) as they approach confluence; the low-passage cells then lose this PA activity after reaching confluence, while the 3T3 cells retain it indefinitely. Even at their peaks, however, the PA activities of the low-passage cells remain well below those of the corresponding virally or spontaneously transformed cells. The PA increases in normal cells are probably a result of PA production rather than of adsorption of secreted PA to the cell surface, or of changes in cell-associated protease inhibitors. The elaboration of PA by normal cells is dependent upon their metabolic activity, such that the level of serum supplementation and the growth phase of the culture directly influence the level of cell-associated PA observed. In addition, there may be a component of serum which exerts a negative control on PA production and which is not an acid-labile protease inhibitor.

scholarly journals CONTACT-INHIBITED REVERTANT CELL LINES ISOLATED FROM SV40-TRANSFORMED CELLS

1973 ◽  
Vol 56 (2) ◽  
pp. 412-428 ◽  
Author(s):  
N. Scott McNutt ◽  
Lloyd A. Culp ◽  
Paul H. Black
Keyword(s):  
Cell Lines ◽  
Transformed Cells ◽  
Cell Periphery ◽  
3T3 Cells ◽  
Heavy Meromyosin ◽  
Thin Sections ◽  
Transformed Cell Line ◽  
Close Relationship ◽  
Loss Of Contact

A comparison is made of the ultrastructure of the cell periphery in three cloned cell lines: untransformed Balb/c 3T3 cells, SV40-transformed Balb/c 3T3 cells, and revertant cells obtained from the transformed cell line by a selection technique utilizing concanavalin A. Both thin-section and surface replication techniques are used for in situ examination of the cell lines. Microfilaments, 70 Å in diameter (called alpha filaments), are abundant in untransformed and revertant cell lines, particularly in the anterior expansions of the cells, which tend to have many microvilli and small pseudopodia. Alpha filaments are diminished in the anterior expansions of transformed cells, which contain large blunt pseudopodia and relatively few microvilli. Surface replicas confirm the impression gained from thin sections that transformed cells have a greater proportion of their cell surface involved in bulging pseudopodia than either untransformed or revertant cells. Since alpha filaments are shown to bind heavy meromyosin and are similar to F-actin, these filaments are thought to be important in cell motility. These observations suggest that a close relationship exists between decreased alpha filaments, bulging pseudopodia, and loss of contact inhibition of movement in transformed cells.

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