A specific effect of external ATP on the permeability of transformed 3T3 cells

Addition of ATP causes a dramatic increase in the rate of p-nitrophenyl phosphate hydrolysis by intact 3T6 and 3T3 cells transformed by polyoma virus and simian virus 40 (SV40). In sharp contrast, untransformed 3T3 cells or secondary mouse embryo fibroblasts, either growing or resting, do not respond to ATP. The activation displays specificity, reversibility and dependence on pH, temperature and ATP concentration. The data suggest that exposure to ATP changes the permeability of transformed cells to p-nitrophenyl phosphate thus revealing an internal, ouabain-insensitive, phosphatase activity.     

Protein degradation in 3T3 cells and tumorigenic transformed 3T3 cells

To study the relation of overall rates of protein degradation in the control of cell growth, we determined if transformation of fibroblasts to tumorigenicity affected their rates of degradation of short- and long-lived proteins. Rates of protein degradation were measured in nontumorigenic mouse Balb/c 3T3 fibroblasts, and in tumorigenic 3T3 cells transformed by different agents. Growing 3T3 cells, and cells transformed with Moloney sarcoma virus (MA-3T3) or Rous sarcoma virus (RS-3T3), degraded short- and long-lived proteins at similar rates. Simian virus 40 (SV-3T3)- and benzo(a)pyrene (BP-3T3)-transformed cells had slightly lower rates of degradation of both short- and long-lived proteins. Reducing the serum concentration in the culture medium from 10% to 0.5%, immediately caused about a twofold increase in the rate of degradation of long-lived proteins in 3T3 cells. Transformed lines increased their rates of degradation of long-lived proteins only by different amounts upon serum deprivation, but none of them to the same extent as did 3T3. Greater differences in the degradation rates of proteins were seen among the transformed cells than between 3T3 cells and some transformed cells. Thus, there was no consistent change in any rate of protein degradation in 3T3 cells due to transformation to tumorigenicity.     

Selection of Revertants of Kirsten Sarcoma Virus Transformed Nonproducer BALB/3T3 Cells

Revertants of Kirsten sarcoma virus transformed nonproducer BALB/3T3 cells (KA31 cells) were isolated after exposing the transformed cells to 5-fluorodeoxyuridine at high cell density, or when suspended in methylcellulose. Revertants were also isolated by treating KA31 cells with the lectin, concanavalin A, which is manyfold more toxic to transformed cells than for normal cells. The revertants resemble BALB/3T3 cells in their morphology and growth characteristics in that they have a low saturation density, fail to grow in 1% calf serum or when suspended in methylcellulose, and cease to synthesize DNA after reaching their saturation density. Infection by murine leukemia virus rescues Kirsten sarcoma virus from only the concanavalin-A-selected variants, though all the revertants are susceptible to infection by leukemia virus. The concanavalin A revertants also become transformed after infection with murine leukemia virus. All the revertants can be transformed by Kirsten sarcoma virus but not by simian virus 40.     

Modification of transplasma membrane oxidoreduction by SV40 transformation of 3T3 cells

Transformation of 3T3 cells by SV40 virus changes the properties of the transplasma membrane electron transport activity which can be assayed by reduction of external ferric salts. After 42 h of culture and before the growth rate is maximum, the transformed cells have a much slower rate of ferric reduction. The change in activity is expressed both by change inK _m andV _max for ferricyanide reduction. The change in activity is not based on surface charge effect or on tight coupling to proton release or on intracellular NADH concentration. With transformation by SV40 virus infection the expression of transferrin receptors increases, which correlates with greater diferric transferrin stimulation of the rate of ferric ammonium citrate reduction in transformed SV40-3T3 cells than in 3T3 cells.     

Heparan sulfates of mouse cells. Analysis of parent and transformed 3T3 cell lines.

Heparan sulfate from the surface of a variety of mouse cells at different cell densities was examined by ion-exchange chromatography. The results of this analysis show that: (1) The heparan sulfate from new isolates of Swiss 3T3 cells transformed by SV40 virus (a DNA tumor virus) elutes from DEAE-cellulose at a lower ionic strength than that from the parent cell type. This finding confirms our earlier observation with an established SV40-transformed cell line (Underhill and Keller, '75) and eliminates the possibility that this change is caused by extended passage in culture. (2) For both parent and transformed 3T3 cells, the heparan sulfates from low and high density cultures were the same as judged by chromatography on DEAE-cellulose. This result demonstrates that the transformation-dependent change which we have observed is independent of cell density. (3) The heparan sulfate from Balb/c 3T3 cells transformed with Kirsten murine sarcoma virus (an RNA tumor virus) elutes from DEAE-cellulose prior to that from parent Balb/c 3T3 cells. This result extends the transformation dependent change in heparan sulfate to the Balb/c 3T3 cell line and to cells transformed with an RNA virus.     

Activation of nuclear chromatin and the release from contact-inhibition of 3T3 cells

A rapid cytophotometric method was developed to measure binding of acridine orange (AO) to the nuclear chromatin of mouse fibroblasts. Contact-inhibited 3T3 cells bound 50–70% less dye than did growing cells in G1 phase of the cell cycle. Release from contact inhibition by fresh serum resulted in stimulated binding of AO within 30 min. No such changes were observed in SV40 virus transformed cells. Differences in AO-binding disappeared at high dye concentration, suggesting that activated cells have a greater affinity to the dye rather than an increased number of binding sites.     

Elevated prostaglandin synthetase activity in methylcholanthrene-transformed mouse BALB/3T3

Cell lines transformed from 3T3 spontaneously, by radiation, or by treatment with chemical carcinogens, polyoma and SV40 virus produce up to 5 times more prostaglandins than their untransformed parent line. Several aspects of prostaglandin biosynthesis by MC5-5 and 3T3 were compared. When stimulated by serum, bradykinin, or thrombin, MC5-5 produced 2-to 5-fold more prostaglandins than 3T3. With the use of cells labeled with radioactive arachidonic acid in their cellular lipids, these higher levels were shown not to be due to increased availability of the prostaglandin precursor, arachidonic acid. Prostaglandin synthetase activity in microsomal fractions prepared from MC5-5 was 6 times higher than that of microsomes of untransformed cells. The increased prostaglandin levels produced by transformed cells therefore appear to be the result of elevated prostaglandin synthetase activity.     

No activation of new initiation points for deoxyribonucleic acid replication in BALB/c 3T3 cells transformed by Kirsten sarcoma virus.

BALB/c 3T3 cells were transformed by Kirsten sarcoma virus, and five clones were isolated in soft agar. Average replicon sizes of the transformed cell lines were estimated by the method of fiber-autoradiography (J. A. Huberman and A. D. Riggs, J. Mol. Biol.32:327-341, 1968) and found to be the same size as the nontransformed 3T3 cells, analyzed in parallel. The results indicate that, unlike simian virus 40 and Epstein-Barr virus, Kirsten sarcoma virus does not activate new initiation points for cellular deoxyribonucleic acid replication in murine sarcoma virus-transformed BALB/c 3T3 cells.     

Difference in transport of leucine in attached and suspended 3T3 cells.

3T3 cells in subconfluent culture take up leucine through a transport system which has a relatively high affinity for leucine (M system). When the culture becomes confluent, the M system is turned off and leucine is transported by another system which has a low affinity for leucine (S system). The M system is reactivated by transferring the cells into subconfluent cultures. In suspension cultures 3T3 cells, initiated from confluent cultures, the M system is not activated and leucine is transported by the S system. In cells suspended from subconfluent culture, the M system continues to operate at a high level for four hours and then is gradually turned off. Tumor virus transformed 3T3 cells (SV3T3 and Py3T3) grow quite well in suspension culture and transport leucine both in monolayer and suspension through a high affinity system, with a high Vmax value. A derivative of 3T3, 3T3/41, which grows in suspension much more slowly than tumor virus transformed 3T3 cells, also takes up leucine through a high affinity transport system both in monolayer and suspension but its Vmax value is lower than that of the transformed cells.     

Four different classes of retroviruses induce phosphorylation of tyrosines present in similar cellular proteins.

Chicken embryo cells transformed by the related avian sarcoma viruses PRC II and Fujinami sarcoma virus, or by the unrelated virus Y73, contain three phosphoproteins not observed in untransformed cells and increased levels of up to four other phosphoproteins. These same phosphoproteins are present in increased levels in cells transformed by Rous sarcoma virus, a virus which is apparently unrelated to the three aforementioned viruses. In all cases, the phosphoproteins contain phosphotyrosine and thus may be substrates for the tyrosine-specific protein kinases encoded by these viruses. In one case, the site(s) of tyrosine phosphorylation within the protein is the same for all four viruses. A homologous protein is also phosphorylated, at the same major site, in mouse 3T3 cells transformed by Rous sarcoma virus or by the further unrelated virus Abelson murine leukemia virus. A second phosphotyrosine-containing protein has been detected in both Rous sarcoma virus and Abelson murine leukemia virus-transformed 3T3 cells, but was absent from normal 3T3 cells and 3T3 cells transformed by various other viruses. We conclude that representatives of four apparently unrelated classes of transforming retroviruses all induce the phosphorylation of tyrosines present in the same set of cellular proteins.     

Elevated prostaglandins synthetase activity in methylcholanthrene-transformed mouse BALB/3T3.

Cell lines transformed from 3T3 spontaneously, by radiation, or by treatment with chemical carcinogens, polyoma and SV40 virus produce up to 5 times more prostaglandins than their untransformed parent line. Several aspects of prostaglandin biosynthesis by MC5-5 and 3T3 were compared. When stimulated by serum, bradykinin, or thrombin, MC5-5 cells labeled with radioactive arachidonic acid in their cellular lipids, these higher levels were shown not to be due to increased availability of the prostaglandin precursor, arachidonic acid. Prostaglandin synthetase activity in microsomal fractions prepared from MC5-5 was 6 times higher than that of microsomes of untransformed cells. The increased prostaglandin levels produced by transformed cells therefore appear to be the result of elevated prostaglandin synthetase activity.     

Competence of soluble cell extracts as microtubule assembly systems. Comparison of simian virus 40 transformed and nontransformed mouse 3T3 fibroblasts.

Soluble cell extracts from simian virus 40 transformed mouse 3T3 fibroblast cells (SV101) and nontransformed mouse BALB/c-3T3 cells were compared as microtubule assembly systems. Extracts from the transformant were found to be at least as competent for microtubule assembly under standard conditions as those from normal cells. This observation proves that the defects in cytoplasmic microtubule skeletons reported in the literature for various transformed cell lines are not due to the loss of integrity of the tubulin molecule itself, but rather to transformation-dependent changes in the regulatory mechanisms controlling in vivo microtubule assembly.     

CONTACT-INHIBITED REVERTANT CELL LINES ISOLATED FROM SV40-TRANSFORMED CELLS : I. Biologic, Virologic, and Chemical Properties

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.     

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

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.     

Effects of angiotensin II and angiotensin II antagonist saralasin on cell growth and renin in 3T3 and SV3T3 cells.

Components of the renin-angiotensin system were studied in established cell culture lines of 3T3 and SV3T3 mouse fibroblasts. The renin content in 3T3 cells was significantly higher than in virus-transformed SV3T3 cells. With time after infection, renin decreased in Simian virus 40 transformed cells, while it increased steadily in mock-infected 3T3 cells. In contrast to renin, angiotensinase activity was higher in SV3T3 cells. Angiotensin II stimulated cell proliferation in 3T3 mouse fibroblasts and decreased their renin content in a dose-related manner. In contrast, saralasin, an angiotensin receptor antagonist, inhibited cell growth in 3T3 and SV3T3 cells and caused an increase of cellular renin concentration. The angiotensin fragments angiotensin (2-8) heptapeptide and angiotensin (4-8) pentapeptide had no effect on cell growth. A significant negative correlation was found between cell proliferation and renin levels in 3T3 and SV3T3 cells irrespective of the treatment. Our results indicate (1) that angiotensin II may be involved in cell growth regulation, (2) that a negative feedback exist between angiotensin II added and intracellular renin content, and (3) that virus infection causes a decrease in intracellular renin synthesis, while non-specific angiotensinase activity is increased under this condition.     

Activation of the Murine Sarcoma Virus Genome After Infection with the Murine Leukemia Virus as Determined by Cell Agglutination

Non-virus-producing NIH/3T3 cells transformed by the murine sarcoma virus are agglutinated by conconavalin A to the same low level as normal NIH/3T3 cells. Infection with the murine leukemia virus greatly increases the agglutination of transformed cells but not that of normal cells. These data suggest that the morphological expression of cell transformation and the surface alterations associated with increased cell agglutination are controlled by the expressions of different sarcoma virus genes.     

In vitro tumoricidal activity of macrophages against virus-transformed lines with temperature-dependent transformed phenotypic characteristics.

The susceptibility of targets to destruction by tumoricidal rat and mouse macrophages was studied with virus-transformed cell lines in which various elements of the transformed phenotype are only expressed at specific temperatures. BHK cells transformed by the ts3 mutant of polyoma virus, rat embryo 3Y1 cells transformed by a temperature-sensitive A cistron mutant of simian virus 40 (SV40) and the ts-H6-15 temperature-sensitive line of SV40-transformed mouse 3T3 cells were killed in vitro by macrophages at both the permissive (33 °C) or nonpermissive (39 °C) temperatures for expression of the transformed phenotype. 3T3, 3Y1 and BHK cells transformed by wild-type SV40 or polyoma virus were also destroyed by tumoricidal macrophages at both 33 and 39 °C, but untransformed 3T3, 3Y1, and BHK cells were not. Thus, transformed cells are killed by macrophages regardless of whether or not they express cell surface LETS protein or Forssman antigen, display surface changes which permit agglutination by low doses of plant lectins, express SV40 T antigen, have a low saturation density, or exhibit density-dependent inhibition of DNA synthesis.     

Insulin receptors in normal and transformed fibroblasts: relationship to growth and transformation

The insulin receptors in normal and transformed lines of mouse Balb/3T3 fibroblasts have been studied. In the normal fibroblasts, the binding of insulin was low in growing cells and increased 2–9 fold in confluent stationary cells. Insulin binding was increased whether growth arrest was due to contact inhibition of growth or serum starvation. When serum-starved cells were stimulated to grow by the addition of fresh serum, insulin binding declined. In cells transformed by simian virus 40, Kirsten, Moloney, and Harvey sarcoma viruses, methylcholanthrene, X rays, or spontaneously, the binding was low, in the same range as growing normal cells. In simian virus 40-transformed cells, insulin binding increased 4 fold as the cells reached higher densities in culture. No relationship to changes in cell size was found. The differences in binding were due to changes in the concentration of the receptors, without changes in their affinity for the hormone.     

Resistance to oncogenic transformation in revertant R1 of human ras-transformed 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.     

Studies on Morphological Transformation of BALB/3T3-Derived Clones by Murine Leukemia Virus

We have described a cell line, UC1-B, derived spontaneously from BALB/3T3 mouse embryo cells, which, unlike the standard BALB/3T3, are morphologically transformed and produce bizarre viral forms in response to murine leukemia virus. Although UC1-B and BALB/3T3 are morphologically similar, and both form contact-inhibited monolayers at confluence, the UC1-B cells are partially transformed because: they grow to a slightly higher saturation density than 3T3 cells, they grow in medium lacking serum growth factors, and they produce tumors in mice. Another clone, 12A-3, derived from BALB/3T3, also transforms and produces bizarre viral forms after infection with murine leukemia virus. Unlike UC1-B cells, the 12A3-8 cells are identical in growth properties to BALB/3T3; therefore, a partially altered morphology is not required for the induction of transformation by murine leukemia virus.