Prostaglandin production by methylcholanthrene-transformed mouse BALB/3T3: Inhibition by cytochalasin B

Cytochalasin B inhibits the production of prostaglandins by serum-, thrombin-, and bradykinin-stimulated MC5-5 cells. The serum-stimulated release of arachidonic acid from cellular phospholipids also is inhibited. Cytochalasin B does not affect the cells' prostaglandin synthetase activity when exogenous arachidonic acid is present. Deacylation of phospholipids may be the step affected by cytochalasin B possibly as a result of disruption of microfilament organization. Colchicine and vinblastine, two drugs that can disrupt microtubule organization, do not inhibit prostaglandin production by cells.     

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.     

Altered kinase C function in transformed BALB/c3T3 cells

Comparative studies of kinase C function were performed in an untransformed (A31) and the benzo[a]pyrene (BPA31), dimethylbenz[a]anthracene (DA31), and Kirsten sarcoma virus (KA31) transformed BALB/c 3T3 mouse fibroblast cell lines. The 80-kDa kinase C dependent phosphoprotein (pp80), an in vivo marker of kinase C activity, was markedly decreased in the transformed cells although the amount of the 80-kDa substrate protein in the BPA31 cells was similar to that in the untransformed A31 cells. Total cell lysate kinase C levels were lower in the transformed cells but this difference could not account for the reduced pp80 phosphorylation. Increased affinity of kinase C for the membrane fraction in the BPA31 cells may account for decreased phosphorylation of pp80.     

A comparison of membrane components of normal and transformed BALB/c cells.

Membrane glycolipids, glycoproteins, and surface proteins of normal and transformed BALB/c cell lines have been compared. Several virally and spontaneously transformed cell lines showed differences in membrane components compared to normal A31 cells. These differences consisted of increased amounts of simpler gangliosides, absence of the large external transformation sensitive (LETS) protein, and the appearance of a major new glycoprotein band of about 105 000 molecular weight. In contrast, the spontaneously transformed cell line that caused the fastest growing tumors in vivo and the most rapid animal death (3T12T) did not have these changes. A31 and 3T12T glycolipid profiles appear similar as did glycoproteins and cell surface proteins detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When Pronase-generated glycopeptides were analyzed by Sephadex G-50 chromatography, and enrichment in faster-eluting species was seen in two killing tumor lines (c5T and 3T12T) compared to A31. Regressing tumor lines (MSC, c5) did not show this change. Isolated membrane glycoproteins yield glycopeptides of different sized after Pronase digestion. In addition, several 3T12T glycoproteins yield glycopeptides that are larger than those from the corresponding glycoproteins of A31 cells. It appears that glycopeptide alterations associated with transformation occur in several membrane glycoproteins.     

Dihydrocytochalasin B. Biological effects and binding to 3T3 cells

Dihydrocytochalasin B (H2CB) does not inhibit sugar uptake in BALB/c 3T3 cells. Excess H2CB does not affect inhibition of sugar uptake by cytochalasin B (CB), indicating that it does not compete with CB for binding to high-affinity sites. As in the case of CB, H2CB inhibits cytokinesis and changes the morphology of the cells. These results demonstrate that the effects of CB on sugar transport and on cell motility and morphology involve separate and independent sites. Comparison of the effects of H2CB, CB, and cytochalasin D (CD) indicates that treatment of cells with any one of the compounds results in the same series of morphological changes; the cells undergo zeiosis and elongation at 2-4 microM CB and become arborized and rounded up at 10-50 microM CB. H2CB is slightly less potent than CB, whereas CD is five to eight times more potent than CB in causing a given state of morphological change. These results indicate that the cytochalasin-induced changes in cell morphology are mediated by a specific site(s) which can distinguish the subtle differences in the structures of the three compounds. Competitive binding studies indicate that excess H2CB displaces essentially all of the high-affinity bound [3H]CB, but, at less than 5 x 10(-5) M H2CB is not so efficient as unlabeled CB in the displacement reaction. In contrast, excess CD displaces up to 40% of the bound [3H]CB. These results suggest that three different classes of high-affinity CB binding sites exist in 3T3 cells: sites related to sugar transport, sites related to cell motility and morphology, and sites with undetermined function.     

Comparison of oxygen consumption rates in minimally transformed BALB/3T3 and virus-transformed 3T3B-SV40 cells

In the recent years, bioenergetics of tumor cells and particularly cell respiration have been attracting great attention because of the involvement of mitochondria in apoptosis and growing evidence of the possibility to diagnose and treat cancer by affecting the system of oxidative phosphorylation in mitochondria. In the present work, a comparative study of oxygen consumption in 3T3B-SV40 cells transformed with oncovirus SV40 and parental BALB/3T3 cells was conducted. Such fractions of oxygen consumption as “phosphorylating” respiration coupled to ATP synthesis, “free” respiration not coupled to ATP synthesis, and “reserve” or hidden respiration observed in the presence of protonophore were determined. Maximal respiration was shown to be only slightly decreased in 3T3B-SV40 cells as compared to BALB/3T3. However, in the case of certain fractions of cellular respiration, the changes were significant. “Phosphorylating” respiration was found to be reduced to 54% and “reserve” respiration, on the contrary, increased up to 160% in virus-transformed 3T3B-SV40 cells. The low rate of “phosphorylating” respiration and high “reserve” respiration indicate that under normal incubation conditions the larger part of mitochondrial respiratory chains of the virus-transformed cells is in the resting state (i.e. there is no electron transfer to oxygen). The high “reserve” respiration is suggested to play an important role in preventing apoptosis of 3T3B-SV40 cells.     

Tumor rejection antigens on BALB3T3 cells transformed by activated oncogenes

The clonal expression of tumor rejection Ag (TRA) was analyzed on nine different clones derived from parental BALB3T3 cells that were transfected with activated H-ras, polyoma middle T (PyMT), c-myc, and v-src oncogenes. It was shown that Bras-h clone, which is an activated H-ras oncogene-induced transformant, expressed TRA as assessed in the transplantation study using syngeneic BALB/c mice. This TRA was not detected on parental BALB3T3 nontransformed cells, suggesting that TRA could be expressed in the BALB3T3 cell transformation. Furthermore, the cross-protection experiments indicated that this TRA was also conferred on other BALB3T3 transformants with high anchorage-independent growth potential such as an activated H-ras transformant Bras-d, and PyMT transformants BMT-f, BnMT-11, BnMT-20, except in the case of one H-ras transformant Bnr-12. In contrast, this TRA was not expressed on the transfectants with little or no anchorage independent growth potential such as a PyMT transfectant BnMT-4, a c-myc transfectant Bmyc-7, and a v-src transfectant Bsrc-7. We developed the mAb BRH19 that could react with TRA+ clones but not with TRA- clones. This mAb makes an immunoprecipitate, which is composed of a 50-kDa single polypeptide chain from Bras-h cell lysate. An injection to mice with this antigen could confer the protection against Bras-h challenge. These data indicate that the 50-kDa putative TRA molecule could be expressed in close association with the cell transformation, irrespective of the introduced oncogenes, and there may exist some regulatory mechanisms rather than individually distinct manners for the expression of TRA.     

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.     

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.     

Tumorigenicity of morphologically distinct transformed foci induced by 3-methylcholanthrene in BALB/c-3T3 cells

4 mm in diameter), invasiveness (smooth vs. invading margins) and other properties (piling vs. spread). In our previous report, we showed that cells from all five types grew in soft agar, transformed normal NIH 3T3 cells and formed foci on normal layer of BALB/c-3T3 cells. In this study, the neoplastic/tumorigenic potential of cells from the five different types of transformed foci was investigated in nude mice. About two million cells from each transformed focus were injected into 4-week-old nude mice. Non-transformed BALB/c-3T3 cells were used as control. The results of this study indicate that all the 45 athymic mice injected with different transformants developed tumors between 2 and 4 weeks after injection. Tumors were not observed in eight mice injected with non-transformed BALB/c-3T3 cells. All tumors were histopathologically confirmed fibrosarcomas. These findings indicate that all five morphologically different foci show tumorigenicity and that any foci of size > or =2 mm regardless of invasiveness and piling could be scored as positive during the cell transformation assay.     

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.     

Cross-reactivity of IgM-secreting B cells from normal BALB/c mice.

Cross-reactive antibodies capable of binding to foreign and self Ag are present in the serum of normal newborn and adult animals. In our work, a chamber ELISA assay was used to quantitate the cross-reactivity of B cells actively secreting Ig in BALB/c mice of different ages. Individual lymphocytes were tested for the production of IgM antibodies capable of binding to a series of four unrelated Ag (DNA, TNP, actin, and OVA). Results indicate that nearly one-quarter of IgM secreting lymphocytes from 6-day-old animals were cross-reactive. This frequency was two- to fourfold higher than that found in adult mice. Very old animals, however, showed a selective increase in the cross-reactivity of anti-DNA (but not anti-TNP) secreting lymphocytes. Evidence from Ag inhibition experiments indicated that low concentrations of soluble Ag could block the binding of polyreactive antibodies, and that approximately one-half of "naturally" cross-reactive B cells produced antibodies capable of binding to three or more unrelated Ag.     

Analysis of hexose transport in untransformed and sarcoma virus-transformed mouse 3T3 cells by photoaffinity binding of cytochalasin B

The effect of simian virus 40 transformation on the hexose transport system in mouse embryo fibroblast Swiss 3T3 cells was examined. The concentration of hexose transporters was estimated by measuring D-glucose-inhibitable cytochalasin B binding. The binding of cytochalasin B to the plasma membranes of simian virus 40-transformed mouse 3T3 cells (SV3T3 cells) was significantly greater than that of 3T3 cells. On the other hand, cytochalasin B binding to the microsomal membranes of SV3T3 cells was decreased, and the total amount of binding to plasma and microsomal membranes was not significantly changed in both cell lines. The electrophoretic analysis demonstrated that both hexose-transporter components of Mr 46 000 and Mr 58 000 affinity labeled were responsible for an increase in the hexose transport by viral transformation. These results suggested that the higher hexose-transport activity of transformed cells is caused by a redistribution of transporter from intracellular membranes to plasma membranes.     

Dissociation by cytochalasin B of movement, DNA synthesis and transport in 3T3 cells.

Cytochalasin B was used as a tool to study the inter-relationships between cell movement, the reinitiated DNA synthesis and the enhanced transport of specific small molecules stimulated by serum in quiescent 3T3 cells. Cytochalasin at concentrations of less than 1 mug/ml inhibits serum-stimulated movement within the monolayer and migration into a wound. Even at ten times this concentration there is little effect on the increase in DNA in the culture, indicating that movement away from neighboring cells is not required for the initiation of DNA synthesis. While DNA synthesis is not inhibited by concentrations of cytochalasin up to 10 mug/ml, the increased thymidine transport which is associated with the onset of the S phase of the cell cycle is inhibited and DNA synthesis cannot be measured by the labelling of nuclei with radioactive thymidine. Cytochalasin has a differential effect on the early transport changes produced by serum addition. Glucose transport is inhibited by low concentrations of the drug (less than 1 mug/ml) while the enhanced uptake of phosphate and uridine is unaffected by a 10-fold increase in concentration. Although the doses of cytochalasin required for 50% inhibition of hexose uptake and of cell movement are the same, no causal relationship between sugar transport and locomotion can be demonstrated. Cytochalasin affects membrane functions in at least two different ways. The drug inhibits the uptake of glucose directly but affects only the S-phase associated increase in thymidine transport.     

Antiproliferative effects of heparin on normal and transformed NIH/3T3 fibroblasts.

We studied the effect of heparin on proliferation and signalling in normal NIH/3T3 fibroblasts, and in cells transformed by different oncogenes. Heparin inhibited the proliferation of normal as well as of v-sis and v-erbB transformed fibroblasts in the presence of serum, but failed to inhibit v-erbB-driven proliferation in serum-starved cultures; under these conditions, heparin inhibited by approximately 50% the proliferation of normal and v-sis- transformed cells. Heparin also inhibited PDGF-induced cell proliferation and inositol lipid turnover in v-sis transformants, but it did not affect PDGF mitogenic signalling in NIH/3T3 fibroblasts.     

Effect of passage in culture on a clone of BALB/c 3T3 cells transformed by simian virus 40.

Most simian virus 40 (SV40)-transformed BALB/c 3T3 clones employed for biochemical studies have been used without regard to passage level. To determine whether virus-induced properties are stable as a function of passage, we have extensively characterized one transformed clone, FNE, which was isolated after SV40 infection BALB/c 3T3 cells in factor-free medium. From the initial testing at passage 5 and for at least 50 subsequent subcultures, the cells stably maintained many transformed growth properties, including high saturation density, morphology, colony formation on contact-inhibited monolayers, tumorigenicity, and synthesis of viral-specific RNA. However, other properties varied as a function of passage. There was a slight decrease in viral genome equivalents per cell from 1.1 copy/cell at passage 5 to 0.7 copies at passage 40. Initially, the cells were negative for all type C virus; however, cells carried at low density for 13 to 20 passages (65 to 100 generations) began to release an endogenous type C virus that then persisted in the culture. Spontaneous release of type C virus did not occur in control BALB/c 3T3 cells carried under identical culture conditions for 90 passages. When the cultures were releasing type C viruses they stained uniformly and brightly positive for SV40 tumor (T) antigen by immunofluorescence, whereas T antigen staining was variable at early passage. These experiments suggest that subtle but perhaps important differences in viral gene expression can occur as a function of passage; they also demonstrate the importance of evaluating the interactions between SV40 and endogenous type C viruses.