Effect of alpha-lipoic acid on 3T3 and 3T3-SV40 fibroblasts: Comparison with N-acetylcysteine

In this study, we have investigated the intracellular level of reduced glutathione, cell-cycle phase distribution, and microfilament and microtubule structures in normal (3T3) and transformed (3T3-SV40) fibroblasts exposed to alpha-lipoic acid (ALA) in concentrations of 0.7–5 mM. It was found that ALA treatment increased the glutathione content in transformed cells, but did not affect its level in normal cells; moreover, it also induced the cell-cycle arrest of 3T3 cells (but not 3T3-SV40 cells) and disrupted actin microfilaments in cells of both lines. The ALA effect was compared to that of N-acetylcysteine (NAC), another antioxidant we examined previously. The findings allow us to assert that each of these antioxidants impacts on distinct target molecules in normal and transformed cells and activates different signal and metabolic pathways in these cells. However, intermediate steps of ALA and NAC action may be common (altered intracellular level of glutathione, reorganization of actin cytoskeleton, etc.).     

Antioxidants-induced rearrangements of actin cytoskeleton in 3T3 and 3T3-SV40 fibroblasts

Effect of antioxidants on actin cytoskeleton in 3T3 fibroblasts and 3T3 fibroblasts transformed with SV40 virus (3T3-SV40 cells) was studied. Antioxidants used were as follows: N-acetyl-L-cysteine (NAC), (-)-2-oxo-4-thiazolidine-carboxylic acid (OTZ), and glutathione in the reduced form (GSH). Both NAC and OTZ are precursors of GSH in the cell, but, in contrast to NAC, OTZ reduces inside the cell forming L-cysteine. The presence of NAC (5-20 mM) in the culture medium of both cell types resulted in loosening of monolayer, fragmentation of stress fibers, and the appearance of amorphous actin structures. As 3T3-SV40 cells contain less actin stress fibers than 3T3 cells, the NAC-induced rearrangements of actin cytoskeleton were stronger in these cells than in 3T3 cells. In contrast to NAC, OTZ (10-20 mM) did not destroy monolayer and did not induce any visible disappearance of stress fibers either in 3T3 or 3T3-SV40 cells. However, in the presence of OTZ, amorphous actin-containing structures were observed in 3T3-SV40 cells. The effect of glutathione on both cell types was similar to that of NAC. The time required for GSH-induced alterations of actin cytoskeleton (about 5 h) was consistent with the increase in the intracellular level of reactive oxygen species (4 h after addition of GSH to the culture medium). Upon removal of the antioxidants from the medium, actin filament structures were reconstructed. However, within 24 h after withdrawal of NAC or GSH, only a partial reconstruction of stress fibers was observed in 3T3 cells. On the contrary, 3T3-SV40 cells demonstrated formation of well-structured actin fibers similar to normal fibroblasts. These results suggest that GSH can act as a pro-oxidant in the absence of oxidative stress.     

N-acetylcysteine reduces transformed 3T3-SV40 fibroblast sensitivity to lysis by natural killer cells

We have shown that 18-20 h cultivation of transformed mouse fibroblasts 3T3-SV40 in the presence of antioxidant, N-acetylcysteine (NAC, 10 mM), did not change their sensitivity to lysis by natural killer (NK) mouse splenocytes. However, in 18-20 h after NAC removal 3T3-SV40 cells demonstrated resistance to NK cell activity. The cytotoxicity index (CI) was reduced up to 4.6 +/- 2.4 % (in comparison with the control value 31.8 +/- 2.4 %) approximating to the value in non-transformed 3T3 mouse fibroblasts. Normal 3T3 cells were resistant to NK action in all experimental conditions (CI varied within 0.7-5.3 %). These results show that NAC can induce partial reversion of transformed phenotype. We suggest that this effect may be due to the NAC-induced modifications of the cell surface and extracellular matrix proteins.     

Activity of matrix metalloproteinases in normal and transformed mouse fibroblasts exposed to antioxidants

The effects of two antioxidants on the activity of matrix metalloproteinases (MMP) secreted by normal (3T3) and transformed (3T3-SV40) mouse fibroblasts were examined. We compared the action of N-acetylcysteine (NAC) and alpha-lipoic acid (ALA) on two gelatinases, MMP-2 and MMP-9. Gel zymography demonstrated that activity of MMP-2 was higher in normal 3T3 cells, whereas, in transformed 3T3-SV40 cells, the MMP-9 activity was higher. NAC treatment for 2–6 h completely suppressed MMP-2 and MMP-9 activity in both cell lines. The inhibitory effect did not depend on NAC concentration within the range of 1–10 mM. ALA (1.2 mM) did not affect the cells very dramatically; it decreased the MMP-2 activity in both types of cells. MMP-9 activity in the presence of ALA was decreased in 3T3 cells and slightly increased in 3T3-SV40 cells. The activity of the membrane bound and intracellular MMP was not changed under the same conditions. In conclusion, the altered activity of MMP in the presence of antioxidant may influence the intracellular signaling and cell functions.     

N-acetylcysteine-induced changes in susceptibility of transformed eukaryotic cells to bacterial invasion

The effect of N-acetylcysteine (NAC) on morphological and physiological properties of "normal" 3T3 and 3T3-SV40 fibroblasts was studied. Incubation of the cells with 10 and 20 mM NAC for 20 h resulted in a reversible increase in the intracellular level of reduced glutathione and disorganization of actin cytoskeleton. Surprisingly, upon removal of NAC, 3T3-SV40 fibroblasts demonstrated formation of well-adhered cells with structured 3T3-like stress-fibers. Neither changes in glutathione levels, nor cytoskeleton disorganization/assembly abolished resistance of 3T3 cells to invasion by the bacterium Escherichia coli A2. On the other hand, pretreatment with NAC converted bacteria-susceptible 3T3-SV40 cells into resistant ones. These results show that NAC can induce partial reversion of transformed phenotype. We suggest that this effect is due to NAC-induced modifications of cell surface proteins rather than to changes in the level of intracellular glutathione.     

Difference in Susceptibility of 3T3 and 3T3-SV40 Cells to Invasion by Opportunistic Pathogens <Emphasis Type="Italic">Serratia grimesii</Emphasis>

Cells isolated from a body are resistant to bacterial invasion but lose the resistance upon immortalization and transformation. In this work, interaction of immortalized 3T3 fibroblasts and their in vitro transformed analog 3T3-SV40 cells with opportunistic bacteria Serratia grimesii was studied in a conventional medium and after incubating the cells with an antioxidant N-acetylcysteine (NAC). The 3T3 cells were shown to be approximately twice less sensitive to S. grimesii infection than similar but virus-transformed 3T3- SV40 cells. Incubation of 3T3 cells with 10 and 20 mM NAC enhanced the invasion 1.6 and 2.5-fold, respectively. Under the same conditions, the invasion of 3T3-SV40 cells by the bacteria was enhanced 2.1 and 2.4- fold. These results show that 3T3 cells are more resistant to invasion by S. grimesii than 3T3-SV40 cells, and the difference is preserved after the cells are exposed to 10 and 20 mM NAC. Among the genes which expression is known to be increased by NAC, a special role plays E-cadherin shown to interact with surface proteins (invasins) of pathogenic bacteria. Incubation of 3T3 and 3T3-SV40 cells with NAC resulted in an increased expression of E-cadherin, which correlates with the increased sensitivity of these cells to invasion. Confocal fluorescence microscopy revealed, for the first time, colocalization of S. grimesii with E-cadherin of 3T3 and 3T3-SV40 cells indicating that E-cadherin can be involved in the penetration of S. grimesii into eukaryotic cells.     

Cyclic AMP-dependent protein kinases from Balb 3T3 cells and other 3T3 derived lines

Cyclic AMP-dependent protein kinase and 3H-cAMP-binding activities were determined in normal Balb 3T3 cells and compared with the same preparations from SV40, chemical, and spontaneous transformants of 3T3 cells. The cytosolic protein kinase activities and protein kinase activity ratios were similar in all cell lines, although when the normal 3T3 cytosol was prepared by homogenization it contained less 3H-cAMP binding activity than the transformed 3T3 cytosols. The Triton X-100 treated particulate fractions from the normal and transformed 3T3 cells contained similar protein kinase and binding activities. The isozymic profile of cAMP-dependent protein kinases was examined by DEAE-chromatography. The 3T3 cells contained only type II isozyme in either cytosolic or membrane fractions. All transformants of the 3T3 cells contained both type I and type II isozymes. Other cell cultures, including chicken embryo fibroblasts, rat kidney cells, and human or calf endothelial cells contained type I and type II isozymes. Binding of the photoaffinity analogue of cAMP, 8-N3 cAMP, to the regulatory subunits of protein kinases in sonicates obtained from Balb 3T3 and SV 3T3 cells followed by separation on SDS polyacrylamide electrophoresis showed that the amount of RII subunit was approximately equal in the two cell lines. RI in Balb 3T3 cells was detectable but in a much lower quantity than in SV 3T3 cells. The cyclic AMP dependent-protein kinases from Balb 3T3 cells appears to be different from SV 3T3 cells by three criteria: 3H-cAMP binding in homogenates, DEAE chromatographic separation of isozymes, and 8-N3 cAMP binding.     

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.     

Contact-activated migration of melanoma B16 and sarcoma XC cells.

During migration, tumour cells interact with neighbouring neoplastic and normal host cells, and such interaction may influence their motile activity. We investigated the effect of homotypic collisions on the motile activity of two tumour cell lines, mouse melanoma B16 and rat sarcoma XC, and nontransformed human skin fibroblasts. It was found that the tumour cells show only limited motile activity when moving as single cells without contact with neighbours. At a higher density of the culture (and also at a greater number of cell to cell contacts) the activation of motility of investigated tumour cells was observed. On the other hand, the normal human skin fibroblasts showed a typical reaction of density-dependent inhibition of motility. The motile activity of tumour cells was not affected by conditioned media and was visibly dependent on a direct physical contact among colliding cells. The activation of cell movement was observed about 40-50 min after the initial contact between tumour cells. Contact-activated migration of neoplastic cells was inhibited by 50 microM verapamil (a selective voltage-gated calcium channel inhibitor) and 10 microM gadolinium chloride (a nonspecific blocker of mechanosensitive ion channels) but not by pertussis toxin. The observation that homotypic collisions among tumour cells strongly increase their motile activity suggests that contact-activated migration may play a significant role in tumour invasion and metastasis.     

Dependence of intracellular alkali-ion concentrations of 3T3 and SV 40-3T3 cells on growth density.

Intracellular contents of potassium and of sodium are determined for 3T3 and SV 40-3T3 cells in dependence of growth density. In parallel, total cell volume and volume of intracellular water is determined for these cells suspended in physiological buffer. Intracellular potassium concentration thus evaluated for suspended 3T3 cells exhibits a sharp decrease at cellular growth densities which lead to density dependent inhibition of cell proliferation. In the case of SV 40-3T3 cells, this drop of potassium concentration with increasing cellular growth density is not observed, which correlates well with the absence of cell density dependent inhibition of cell growth in the transformed cell line. These results support the notion that processes of stimulation of quiescent 3T3 cells or of cell density dependent inhibition of their proliferation are mediated by processes including changes of potassium transport characteristics leading to increase or decrease respectively of their intracellular potassium concentration. Furthermore, these and other results suggest, that a difference between normal and transformed cells most relevant to their different proliferation behaviour might reside in different transport characteristics for potassium of the plasma membranes of these cells.     

Mechanosensitive ion channels

The article concentrates on the concepts of mechanosensitive ion channels that are present in practically all cells of an organism. Considered are kinetic scheme and activation principles of mechanic-sensitive ion channels. The forces affecting those channels are discussed in detail. The qualities of the channels in lipid monolayer, bilayer and real cell membrane are under consideration. Discussed are various models that analyze possibilities of channel opening depending on the membrane tension. Under discussion are the data received from studying single channels, currents in whole-cell configuration and cloned channels built into bilayer, liposomes and membrane blebs. Problems of transmitting mechanic energy to the channel through the bilayer and through the cytoskeleton are investigated. Inhibitors and activators of mechanosensitive ion channels are mentioned and their effects are considered. The functional classification of mechanosensitive ion channels is given. Described are cation SACs, potassium SACs, Ca(2+)-sensitive and Ca(2+)-insensitive SACs, anion SACs, nonselective SACs and SICs. It is proved that mechanosensitive ion channels can produce considerable currents enough to change the cell electrogenesis.     

An alteration in tubulin expression detected in SV40 transformed 3T3 cells

Tubulin expression was analysed in normal and simian virus-40 (SV40) transformed 3T3 cells by two-dimensional polyacrylamide gel electrophoresis and immunoblotting studies using monoclonal antibodies raised to alpha- and beta-tubulin subunits. The ratio of alpha- to beta-tubulin recognised was calculated for both cell lines and found to shift from 2.50 in normal cells to 0.52 in virally transformed cells. beta-Tubulin was thereby shown to be the predominant subunit in SV40-transformed 3T3 cells in contrast to normal 3T3 cells.     

Antioxidant action on the level of reactive oxygen species in normal and transformed fibroblasts

The level of reactive oxygen species (ROS) in normal (3T3) and transformed (3T3-SV40) murine fibroblasts treated with antioxidants for 15 min was studied using a carboxy-H₂DCFDA fluorescent probe. It was shown that N-acetylcysteine (NAC) decreased the ROS level in both cell types. Antioxidant alpha-lipoic acid (ALA) and its reduced form dihydrolipoic acid (DHLA) caused a pro-oxidant effect. ALA and DHLA in the concentration range from 0.1–1.25 mM increased the ROS level in a dose-dependent manner in both cell types. The ability of ALA and DHLA to activate hydrogen peroxide production is discussed.     

N-acetylcysteine reduces susceptibility of transformed and embryonic cells to lytic activity of natural killer cells

Changes in the sensitivity of several transformed and embryonic cells to the lytic activity of natural killers (NK) due to N-acetylcysteine (NAC) has been studied. We found that epidermoid carcinoma A431 cells and murine hepatoma MH22a cells, as well as the transformed mouse fibroblasts 3T3-SV40 treated with 10 mM NAC that we investigated previously normalized their phenotype to the various extent. Like normal cells these cells exposed to NAC are not recognized and destroyed by NK. Murine embryonic fibroblasts (MEFs) similar to transformed cells were destroyed by NK. MEFs treated with 10 mM NAC lost their susceptibility to NK, as did transformed cells. The loss of cell sensitivity to NK cytolytic activity was accompanied by the reorganization of the actin cytoskeleton and generation of well-pronounced stress-fibers.     

Reorganization of actin cytoskeleton in 3T3-SV40 cells and their sensitivity to lytic activity of natural killer cells

The goal of the present work was to find out whether the actin reorganization of 3T3-SV40 cells affects their sensitivity to the activity of natural killer (NK) cells. The effects of N-acetylcysteine (NAC) and the inhibitor of actin depolymerization, latrunculin B on both cellular parameters were studied. Experiments with NAC demonstrated that the sensitivity of 3T3-SV40 cells to the NK cell activity remained unchanged with disordered microfilaments, but decreased upon the appearance of structured stress-fibers. With respect to latrunculin B action, the opposite conclusion has been drawn, which is that the more microfilaments disorganization in the presence of latrunculin B, the lower the 3T3-SV40 sensitivity to lysis by NK cells. These facts suggest that relations between microfilament integrity in 3T3-SV40 cells and their sensitivity to NK cells are rather independent, which confirms our previous conclusion (Gamaley et al., 2006). A decrease in the 3T3-SV40 sensitivity to the NK cell activity accompanied by actin reorganization resulting from the influence of both latrunculin B and NAC suggests changes in the cellular surface that ultimately lead to the inactivation (or loss) of molecules that are activating signals to NK cells.     

P2Y purinoceptors in normal NIH 3T3 and in NIH 3T3 overexpressing c-ras

The ability of purinergic agonists to induce Ca2+ responses has been tested in two lines of murine fibroblasts: normal NIH 3T3 fibroblasts and NIH 115.14, a clone expressing high levels [1] of the c-ras protooncogene. Both kinds of cells are responsive to ATP in the range 1 microM-1 mM; ADP and ATP gamma S are almost as potent as ATP, while AMP is unable to elicit a response. Ca2+ measurements performed in single cells by image analysis show great variability among cells but in each individual responding cell the Ca2+ rise occurs in an all-or-none fashion. The transient Ca2+ response does not depend on influx from the extracellular medium. Electrophysiological experiments reveal the activation of an outward current (at -50 mV) by ATP, probably due to Ca(2+)-activated K+ channels, confirming the absence of a substantial Ca2+ influx. Finally, stimulation by ATP produces a small but significant increase in the production of inositol phosphates. These results indicate that these cell lines possess purinergic receptors which are not integral membrane channels and which are coupled to InsP3 formation and may be therefore classified as P2Y.     

Properties of particle movement in the plasma membrane of 3T3 mouse fibroblasts

The effects of cytochalasin B, vinblastine and temperature on particle movement in the plasma membrane of several 3T3 mouse fibroblast lines were investigated. Preincubation of normal 3T3 cells for 24 h in 5–10 μg/ml cytochalasin B had no effect on the mean square relative displacement of marker particles in the membrane (motion constant), but preincubation for 4 h in 40 μg/ml vinblastine reduced the value of this constant by 70%. A 10 °C decrease in temperature decreased the motion constant in normal cells (Q₁₀ = 4) more than in virus-transformed 3T3 cells (Q₁₀ = 1.8). Interpreting the motion constant of the particles as an expression of the viscosity of the membrane material, values of 3 poise for normal 3T3 cells and 6 poise for the transformed cells are obtained for 37 °C and pH = 7.4.A method is suggested to quantitate aggregation of particles on the surface of cells. When this method is applied to gold particles on 3T3 cells, disaggregation of particles is seen to behave as an unordered process, whereas aggregation appears to express cellular control. This consideration and the effect of vinblastine indicate that the interpretation of particle movement as Brownian movement in a viscous membrane material does not cover all phenomena observed.The membrane movement of the flat revertant SVF1 101 [1] was investigated. This cell line occupies an intermediary position between normal 3T3 mouse fibroblasts and the polyoma and SV40 transformed 3T3 cell lines as judged by growth properties. Its membrane movement was found to occupy an intermediary position between the membrane movements of these cell lines, too.     

Calmodulin and Ca2+ in normal and transformed cells

Numerous lines of evidence implicate calcium and calmodulin (CaM) as regulators of cell growth and functional differentiation. In light of this evidence, several studies of the possible involvement of the CaM system in cellular transformation by RNA and DNA tumor viruses have been carried out. This paper summarizes the evidence linking calcium and CaM to the regulation of cell growth and critically examines the evidence that increases in CaM levels occur in transformed versus normal cells. A nontraumatic method for synchronizing both normal and transformed chick fibroblasts is presented. This method is utilized in a comparison of CaM level throughout the cell cycle of Rous sarcoma virus transformed and normal chick embryo fibroblasts. These studies best support the hypothesis that the observed differences in CaM levels between transformed and normal cultures under optimal growth conditions may largely reflect differences in the proportion of cells in a dividing versus a nondividing state.     

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.