Potassium transport in normal and transformed mouse 3T3 cells

The components of unidirectional K influx and efflux have been investigated in the 3T3 cell and the SV40 transformed 3T3 cell in exponential and stationary growth phase. Over the cell densities used for transport experiments the 3T3 cell goes from exponential growth to density dependent inhibition of growth (4 × 10⁴ to 4 × 10⁵ cell cm^?2) whereas the SV40 3T3 maintains exponential or near exponential growth (4 × 10⁴ to 1 × 10⁶ cell cm^?2). In agreement with previous observations, volume per cell and mg protein per cell decrease with increasing cell density. Thus, transport measurements have been expressed on a per volume basis. Total unidirectional K influx and efflux in the 3T3 cell is approximately double that of the SV40 3T3 cell at all cell densities investigated. Both cell types have similar volumes initially and show similar decreases with increasing cell density. Thus, in this clone of the 3T3 cell SV40 transformation specifically decreases unidirectional K flux. The magnitude of the total K flux does not change substantially for either cell line during transition from sparse to dense cultures. However, the components of the K transport undergo distinct changes. Both cell lines possess a ouabain sensitive component of K influx, presumably representing the active inward K pump. Both also possess components of K influx and efflux sensitive to furosemide. The data suggest this component represents a one-for-one K exchange mechanism. The fraction of K influx mediated by the ouabain sensitive component is reduced to one half its value when exponential versus density inhibited 3T3 cells are compared (63% versus 31% of total influx). No comparable drop occurs in the SV40 3T3 cell at equivalent cell densities (64% versus 56% of total influx). Thus, the pump mediated component of K influx would appear to be correlated with growth. In contrast, the furosemide sensitive component represents approximately 20% of the total unidirectional K influx and efflux in both cell lines in sparse culture. At high cell densities, where growth inhibition occurs in the 3T3 cell but not the SV40 3T3, the furosemide sensitive component doubles in both cell lines. Thus, the apparent K-K exchange mechanism is density dependent rather than growth dependent.     

Ammonia effects in cultures of normal and transformed 3T3 cells

3T3 and SV-40 transformed 3T3 mouse fibroblasts were cultured in media with serum and antibiotics plus ammonia (NH₃ z NH₄⁺) added as NH₄C1. Both cell lines cultured without added ammonia showed normal morphology and multiplication even though ammonia in the medium at the end of the culture period ranged from 35 to 48 μg/ml. Ammonia concentrations being significantly higher in media removed from cells at the end of the culture period than in media incubated identically without cells, verified that cells released substantial quantities of ammonia in addition to components of the medium which underwent spontaneous breakdown. Both cell lines showed changes in morphology and highly significant reductions in cell multiplication which increased progressively as the concentration of added ammonia on the initial day of culture was increased to 35μg/ml. Control 3T3 cultures released significantly greater quantities of ammonia per cell than control cultures of transformed cells but their multiplication was more adversely affected by added ammonia. There were downward shifts in pH of the culturing medium for both cell lines as culture age increased at all concentrations of added ammonia, However, significant reductions in cell multiplication resulted from additions of ammonia that did not produce significant changes in extracellular pH. The data show that studies upon the effects of pH of the medium on cultured cells require control of ammonia concentrations.     

Expression of microtubule networks in normal cells, transformed cells, and their hybrids

Microtubules play an important role in several cellular functions including cellular architecture and chromosome movement in cell division. Tubulin which polymerizes to form mictobules can be purified to homogeneity and used to raised antisera. Antisera prepared against porcine or chicken tubulin reacts well with mammalian tubulin. We have examined normal and transformed cells of mouse and human origin for microtubules by indirect immunofluorescence methods. Extensive networks of microtubules (MN) are easily detectable in normal and some transformed cells. The fixation procedure employed and the morphology and the cellular attachment properties seem to determine the ease of detection of MN in these cells. Cells derived from tumors and exhibiting several transformed phenotypes contained MN comparable to those of normal cells. Hybrids between transformed mouse cells and normal human cells were examined. They showed a variability in morphology, but all contained MN. These hybrids exhibited several transformed phenotypes. We conclude that in the cell lines we have examined there is no correlation between the transformed phenotypes and the organization of tubulin.     

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.     

Actin content and organization in normal and transformed cells in culture

The amount of actin and total protein per cell in normal rat kidney (NRK) cells in culture is initially high in very low density cultures, but rapidly decreases as the cells come into contact in higher density cultures. In a viral transformant of NRK (442), the level of actin and total protein does not change significantly from low to high density cultures. NRK cells, which are flattened against the substrate, have prominent bundles of actinlike microfilaments in the basal cytoplasm adjacent to the substrate. 442 cells, which adhere poorly and are more spherical in shape, lack well-organized basal microfilament bundles, but may display microfilament bundles in cytoplasmic processes extending from the cell body. The percentage of insoluble actin is less than 20% in both cell lines, and 442 cells consistently contain smaller amounts than NRK cells.     

High-affinity cytochalasin B binding to normal and transformed BALB/3T3 cells.

To study the molecular basis of changes in sugar uptake rate in cultured mouse fibroblasts with different physiological states, we have measured the high affinity binding of [3H] cytochalsin B, a potent sugar transport inhibitor, to actively growing and contact inhibited Balb/3T3 cells as well as to 3T12 and SV3T3 cells. Binding was the same whether the cells were detached from dishes with EDTA or trypsin. The amount of drug bound to intact cells measured with a centrifugation assay was essentially the same as that bound to cell sonicates measured with equilibrium dialysis. Cytochalasin B binding to intact cells was extremely rapid and reversible over a wide range of drug concentrations, and was not affected by 0.1 M D--glucose in the assay medium. Actively growing and contact inhibited 3T3 cells had a similar number of high affinity cytochalasin B binding sites per cell, while 3T12 and SV3T3 cells had one third to one fourth the number of sites per cell. However, the number of sites per mug cellular protein appeared to be similar for cells in all of the physiological states examined.     

Pyridine nucleotide levels as a function of growth in normal and transformed 3T3 cells.

The levels of NAD (NAD⁺ + NADH) and NADP (NADP⁺ + NADPH) and their redox states were measured as a function of growth in 3T3 mouse fibroblasts which exhibit density-dependent inhibition of growth and SV40 (simian virus #40)-transformed 3T3 cells (SVT2) which have lost this property. The levels were related to cell numbers, protein content, and rates of DNA synthesis. At corresponding cell densities, 3T3 cells contain approximately twice as much total protein as SVT2 cells. The levels of NAD relative to total cellular protein are density dependent in both 3T3 and SVT2, increasing with increasing cell density. Over a 30-fold range of cell densities, the NAD levels in 3T3 increase 2.4-fold, while the levels in SVT2 increase 1.6-fold. The levels of NAD are very similar in dividing 3T3 and SVT2 cells at corresponding cell densities; however, a marked increase in the levels of NAD is observed in 3T3 cells, but not in SVT2 cells, at cell densities just prior to where 3T3 cells enter density-dependent inhibition of growth. This increase in NAD levels is correlated with the cessation of DNA synthesis. The NAD pools are 15–25% NADH for 3T3 and 5–15% NADH for SVT2. NADP levels relative to protein in 3T3 and SVT2 are less density dependent, with overall increases of 1.3- and 1.2-fold, respectively, observed over the range of cell densities examined. NADP levels relative to protein are nearly twice as high in SVT2 cells as in 3T3 cells of corresponding cell densities. The NADP pools are approximately 70–80% NADPH in both cell types.     

Gene expression in normal and virally transformed mouse 3T3B and hamster BHK21 cells

Cell lines 3T3B (mouse), 3T3B-SV40, BHK21 (hamster) and BHK21 polyoma virus (PyY) were labelled with [³⁵S]methionine under conditions in which 500–600 cpm were incorporated per cell during a 20 h incubation period. Two-dimensional gel electrophoresis analysis of the total [³⁵S]methionine-labelled polypeptides from 200–300 cells followed by fluorography revealed about 500 acidic (isoelectric focusing, IEF) and 150 basic polypeptides (non-equilibrium pH gradient electrophoresis, NEPHGE) whose position could be reproducibly assessed. Counting of 33 abundant acidic polypeptides present in both 3T3B and 3T3B-SV40 revealed significant changes in the relative proportion of ten of them. Seven, including the subunit of the 100 Å filaments ‘fibroblast type’ (55K) (1.1% in 3T3B; 0.6% in 3T3B-SV40), three cytoarchitectural proteins and three soluble proteins, corresponded to a decrease of 40% or more in the radioactivity of the spots in transformed cells, and only in three cases was there a significant increase in radioactivity of polypeptides in 3T3B-SV40 cells. Among the polypeptides that show less than 40% variation we have identified total actin (42K) (13% of total label in 3T3B; 10% in 3T3B-SV40), α- and β-tubulin (55K) (1.6% of total label in 3T3B; 2% in 3T3B-SV40), eleven polypeptides present in Triton skeletons, and nine soluble proteins. We have also observed 25 obvious changes in polypeptide intensities (16 acidic and 9 basic) but these were not quantitated. Only three polypeptides were found in transformed cells that were not detected in normal cells. One of these corresponded to the large T antigen and the other two to Triton-soluble proteins of a molecular weight in the range of 52–54K. Similar quantitative studies on the hamster BHK21/BHK21PyY pair confirmed at least the major observations made in 3T3B and 3T3B-SV40.     

Quantitative analysis of microtubule orientation in interdigitated leaf pavement cells

Leaf pavement cells are shaped like a jigsaw puzzle in most dicotyledon species. Molecular genetic studies have identified several genes required for pavement cells morphogenesis and proposed that microtubules play crucial roles in the interdigitation of pavement cells. In this study, we performed quantitative analysis of cortical microtubule orientation in leaf pavement cells in Arabidopsis thaliana. We captured confocal images of cortical microtubules in cotyledon leaf epidermis expressing GFP-tubulinβ and quantitatively evaluated the microtubule orientations relative to the pavement cell growth axis using original image processing techniques. Our results showed that microtubules kept parallel orientations to the growth axis during pavement cell growth. In addition, we showed that immersion treatment of seed cotyledons in solutions containing tubulin polymerization and depolymerization inhibitors decreased pavement cell complexity. Treatment with oryzalin and colchicine inhibited the symmetric division of guard mother cells.     

Comparative analysis of pre-replication complex proteins in transformed and normal cells

This study examines the abundance of the major protein constituents of the pre-replication complex (pre-RC), both genome-wide and in association with specific replication origins, namely the lamin B2, c-myc, 20mer1, and 20mer2 origins. Several pre-RC protein components, namely ORC1-6, Cdc6, Cdt1, MCM4, MCM7, as well as additional replication proteins, such as Ku70/86, 14-3-3, Cdc45, and PCNA, were comparatively and quantitatively analyzed in both transformed and normal cells. The results show that these proteins are overexpressed and more abundantly bound to chromatin in the transformed compared to normal cells. Interestingly, the 20mer1, 20mer2, and c-myc origins exhibited a two- to threefold greater origin activity and a two- to threefold greater in vivo association of the pre-RC proteins with these origins in the transformed cells, whereas the origin associated with the housekeeping lamin B2 gene exhibited both similar levels of activity and in vivo association of these pre-RC proteins in both cell types. Overall, the results indicate that cellular transformation is associated with an overexpression and increased chromatin association of the pre-RC proteins. This study is significant, because it represents the most systematic comprehensive analysis done to date, using multiple replication proteins and different replication origins in both normal and transformed cell lines.     

The use of immunostaining to quantify x-ray and heat-induced multiple microtubule organizing centers in normal and transformed cells

Microtubule organizing centers (MTOC) in control, irradiated and heated C3H 10T1/2 mouse embryo cells and two radiation-transformed sublines, R1 and R25, were made visible by indirect immunofluorescence using antibody against tubulin. The MTOC were reformed by 5-min incubation in fresh medium after the microtubules were depolymerized with nocodazole. The R1 line had a different distribution of MTOC/cell than the parent 10T1/2 line or R25, which had similar distributions. After irradiation, multiple MTOC appeared in the normal and radiation-transformed cells irradiated to 10 Gy and incubated for 24 or 48 h. The multiple foci of microtubule reformation in the irradiated cells indicate that radiation damage is expressed in structural elements in the cytoplasm. After heat treatment of the three cell lines (43 degrees C for 93 min and 45 degrees C for 25 min), the MTOC were disrupted and many cells did not have visible organizing centers at 24 or 48 h, while others had a large number of small centers of microtubule reformation. The distribution of MTOC/cell seen in R25 cells after the treatment had similar patterns to those of the 10T1/2 line rather than to those of the other radiation-transformed line, R1. Thus, the radiation or heat response seen in the MTOC is not dependent upon cell transformation.     

Traction force microscopy of migrating normal and H-ras transformed 3T3 fibroblasts

Mechanical interactions between cell and substrate are involved in vital cellular functions from migration to signal transduction. A newly developed technique, traction force microscopy, makes it possible to visualize the dynamic characteristics of mechanical forces exerted by fibroblasts, including the magnitude, direction, and shear. In the present study such analysis is applied to migrating normal and transformed 3T3 cells. For normal cells, the lamellipodium provides almost all the forces for forward locomotion. A zone of high shear separates the lamellipodium from the cell body, suggesting that they are mechanically distinct entities. Timing and distribution of tractions at the leading edge bear no apparent relationship to local protrusive activities. However, changes in the pattern of traction forces often precede changes in the direction of migration. These observations suggest a frontal towing mechanism for cell migration, where dynamic traction forces at the leading edge actively pull the cell body forward. For H-ras transformed cells, pockets of weak, transient traction scatter among small pseudopods and appear to act against one another. The shear pattern suggests multiple disorganized mechanical domains. The weak, poorly coordinated traction forces, coupled with weak cell-substrate adhesions, are likely responsible for the abnormal motile behavior of H-ras transformed cells.     

Effects of microtubule modulators on HIV-1 infection of transformed and resting CD4 T cells

Previous studies have observed fluorescently labeled HIV particles tracking along microtubule networks for nuclear localization. To provide direct evidence for the involvement of microtubules in early steps of HIV infection of human CD4 T cells, we used multiple microtubule modulators such as paclitaxel (originally called taxol; 1 μM), vinblastine (1 and 10 μM), colchicine (10 and 100 μM), and nocodazole (10 and 100 μM) to disturb microtubule networks in transformed and resting CD4 T cells. Although these drugs disrupted microtubule integrity, almost no inhibition of HIV-1 infection was observed. Our results do not appear to support an essential role for microtubules in the initiation of HIV infection of CD4 T cells.     

Differential adaptive response to hyperosmolarity of 3T3 and transformed SV3T3 cells

Both 3T3 and simian virus 40-transformed 3T3 (SV3T3) cells were used to investigate differences in population kinetics, protein synthesis, monovalent ion levels, and amino acid accumulations between normal and transformed cells exposed to hyperosmolarity at 0.5 Osm. Under similar culture conditions, SV3T3 cells were found to be more sensitive in their proliferative response than normal cells to the hyperosmolar treatment. In the normal 3T3 cells, the increase in transport of amino acids was less sustained and was associated with higher levels of accumulated amino acids. The equilibrium distribution of intracellular monovalent cations and the rate of protein synthesis also returned faster to baseline values in the normal cells than in the transformed cells. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) analysis revealed the induction of a 69-kDa polypeptide in the 3T3 cells but not in the SV3T3 cells after exposure to hyperosmolarity. On electrofocusing and relative mass analysis, this polypeptide closely migrated with the 70-kDa heat shock protein (hsp) family, although it was unrelated immunologically to the inducible 72-kDa hsp.     

Cell cycle dependent modulations of the surface membrane of normal and SV40 virus transformed 3T3 cells

Agglutinability with Concanavalin was studied as function of cell cycle transition in normal and SV40 virus transformed 3T3 cells. In synchronized cultures of normal cells, agglutinbility was high during mitosis and disappeared rapidly. Agglutinability of transformed cells remained high in G₁ phase but diminished gradually upon entering S phase and reached minimum in G₁ phase. Decreased agglutinability a the end of the cell cycle was also observed in synchronous SV3T3 cultures by a combined technique of haemadsorption and density gradient centrifugation. In normal 3T3 cells, similar variations in agglutin ability during interphase could not be observed.     

Enucleation of normal and transformed cells

A quantitative analysis based on centrifugal force requirements for enucleation was developed to examine the response of a number of untransformed and transformed cell lines to cytochalasin mediated enucleation. Examination of the extent of cell enucleation as a function of centrifugal force resulted in a series of response curves demonstrating that enucleation g force requirements varied between Balb/c 3T3, Swiss 3T3, and Kirsten sarcoma virus transformed Balb/c 3T3 (3T3-K). A four times greater centrifugal force was required to reach 50% enucleation for transformed Balb/c 3T3-K when compared to Swiss 3T3. A qualitative correlation could be observed between ease of enucleation and the existence of a well-formed stress fiber network. A comparison of cytochalasin B and D suggested that cytochalasin D was far more effective in the enucleation of transformed cells. Experiments with 2-deoxyglucose and monensin provided evidence that decreasing cellular ATP levels, either directly or potentially by uncoupling ion transport from ATP generation, can decrease the efficiency of enucleation. It is suggested that the organization of the cytoskeleton is affected by the altered cellular ATP levels which can affect the centrifugal requirements of enucleation.     

Effects of bovine platelet-poor plasma on the proliferation of normal and transformed BALB/c 3T3 cells

Summary Platelet-poor plasma, as well as autologous platelet-rich serum, was prepared from freshly-drawn bovine whole blood. Bovine platelet-poor plasma had properties similar to those previously decribed for human platelet-poor plasma; e. g., it would (a) support the growth of virally transformed but not normal BALB/ c 3T3 cells, (b) act synergistically with either partially purified platelet-derived growth factor or fibroblast growth factor to initiate cell replication in quiescent 3T3 cells, and (c) act sequentially with platelet-derived growth factor to initiate 3T3 replication. It appears that bovine serum contains both competence and progression factors and that stimulation of fibroblasts with bovine serum involves at least two sequential stages analogous to those described for stimulation with human serum.     

Quiescent SV40 virus transformed 3T3 cells in culture

Serum counteracts low nutrient concentrations in the culture medium in SV40 virus transformed 3T3 (SV3T3) cells. The transport of [³H]-leucine into TCA soluble material in SV3T3 cells is stimulated by serum and inhibited by But₂-cAMP. When SV3T3 cells are cultured in low leucine concentrations (? 8 × 10^?6 M), the cell's morphology is similar to the one of cells incubated in complete medium in the presence of But₂-cAMP and cells become quiescent. Cells become arrested throughout the cell cycle. The results suggest that the mechanism by which But₂-cAMP inhibits growth of SV3T3 cells is by inhibiting the transport of leucine in SV3T3 cells.     

VSV replication in normal and transformed T cells, an assay for T suppressor cell activation

An infectious center viral plaque assay has been utilized to quantitate activated T suppressor (Ts) cells. This assay is based on two observations. Namely, resting T cells do not serve as good replicative hosts for many viruses, including vesicular stomatitis virus (VSV), and that Ts cells can be enriched by their ability to bind to antigen-coated dishes. Our data show that Ts cells specific for either the TNP hapten or for dextran will replicate VSV upon antigenic and/or mitogenic activation, whereas resting Ts and hapten-specific B cells are less efficient in this process. This system will now allow the direct quantitation of Ts cells and their activation properties.