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.     

Differential methylation of mitochondrial and nuclear DNA in cultured mouse, hamster and virus-transformed hamster cells. In vivo and in vitro methylation

Information has been lacking as to whether mitochondrial DNA of animal cells is methylated. The methylation patterns of mitochondrial and nuclear DNAs of several mammalian cell lines have therefore been compared by four methods: (1) in vivo transfer of the methyl group from [methyl-³H]methionine; (2) in vivo incorporation of [³²P]orthophosphate and a combination of (1) and (2); (3) in vivo incorporation of [³H]deoxycytidine; (4) in vitro methylation of DNAs with ³H-labeled S-adenosylmethionine as methyl donor and DNA methylase preparations from L cell nuclei. The cell lines were mouse L cells, $\text{BHK21}\text{C13}$, $\text{C13}\text{B4}$ (baby hamster kidney cells transformed by the Bryan strain of Rouse sarcoma virus), and PyY (BHK cells transformed by polyoma virus). DNA bases were separated chromatographically, using 5-methylcytosine, 6-methylaminopurine and, in some cases, 7-methylguanine as markers.Mitochondrial DNA was found to be significantly less methylated than nuclear DNA with respect to 5-methylcytosine in all cell types studied and by all methods used. The relative advantages and disadvantages of each method have been discussed. The level of 5-methylcytosine in mitochondrial DNA as compared with that in nuclear DNA was estimated as one-fourth to one-fourteenth in various cell lines. The estimated 5-methylcytosine content per circular mitochondrial DNA molecule (mol. wt 10 × 10⁶) was about 12 methylcytosine residues for L cells and 24, 30 and 36 methylcytosine residues for BHK, B4 and PyY cells, respectively. Relative to cytosine residues, the estimate was one 5-methylcytosine per 500 cytosine residues of mitochondrial DNA and one 5-methylcytosine per 36 cytosine residues of nuclear DNA from L-cells. The values for methylcytosine of mitochondrial DNA are presumed to be maximal. PyY cells as compared with other cells had the highest methylcytosine content of both mitochondrial and nuclear DNA as estimated by method (3). No methylation of nuclear DNA was observed in confluent L cells.Evidence for the presence of DNA methylase activity associated with mitochondrial fractions was obtained. This activity could be distinguished from other cellular DNA methylase activity by differential response to mercaptoethanol. Radioactivity from ³H-labeled S-adenosylmethionine was found only in 5-methyl-cytosine of DNA.     

Direct microinjection of rabbit globin mRNA into mouse 3T3 cells. Analysis of the polypeptides synthesized in vivo

3T3 cells grown attached to 9 mm² coverslips have been microinjected in the cytoplasm with total rabbit globin mRNA and the polypeptides synthesized after injection have been labelled with [³⁵S]-methionine under conditions in which the product of as few as 100 cells could be analysed by high resolution two-dimensional gel electrophoresis followed by 10 days' fluorography. Microinjection of rabbit globin mRNA results in the synthesis of a basic polypeptide of mol. wt 15 K that is not present in control cells, and that co-migrates with purified [³H]leucine-labelled globin as determined by high resolution two-dimensional gel electrophoresis (NEPHGE). Visual inspection of the fluorograms revealed that the injection of globin mRNA (up to 14000 molecules/cell) does not alter significantly the relative intensity of the major acidic (IEF) and basic (NEPHGE) polypeptides synthesized by the cells.     

Inhibition of spontaneous aggregation by wheat germ agglutinin in suspensions of BALB/c-3T3 and BHK21 tissue culture fibroblasts

A quantitative method of measuring cytoaggregation based on the Coulter electronic cell counter has been applied to the agglutination of BALB/c-3T3 and BHK21 tissue culture fibroblasts by wheat germ agglutinin. When agglutinin is added to transformed or trypsinized cell suspensions high aggregation rates are seen, and the results obtained are in close agreement with the well-known sensitivity of transformed cells to agglutination by lectins.In the absence of agglutinin, a small but reproducible amount of spontaneous aggregation can also be detected. It is related in some way to growth, since it is absent in suspension prepared from confluent (density-inhibited) cultures and is induced by either transfer to low density, additional serum, or transformation by viruses. Under conditions favouring growth, both BALB/c-3T3 and BHK21 cells show aggregation indices close to 25, corresponding to 10% of the maximum aggregation rate seen.This spontaneous aggregation is susceptible to inhibition by agglutinin. Inhibition occurs at low concentration (about 10 μg/ml) and aggregation rates thus pass through a minimum as the concentration of agglutinin is increased. Among the four different cell lines studied, sensitivity to inhibition is inversely related to agglutination. Thus 3T3 cells, which are barely agglutinated by 1 mg/ml of agglutinin, show 90% inhibition; polyoma virus-transformed BHK cells, which are agglutinated by 10 μg of agglutinin, show no inhibition at all.It is suggested that the agglutination of transformed cells is a consequence of their failure to respond to an inhibitory effect exerted by lectins upon an intrinsic adhesive property of the cell surface.     

Characterization of the DNA-cellulose-binding proteins from Escherichia coli K 12

The various [35S]DNA-binding proteins present in lysates of Escherichia coli K 12 cells have been analyzed by means of two-dimensional SDS-polyacrylamide gel electrophoresis. The proteins were isolated by the DNA-cellulose technique and eluted by increasing concentrations of NaCl (0.15, 0.4, 0.6 and 2 M). Only 2% of the total 35S radioactivity in the lysate became bound to the DNA-cellulose column. A total of 237 polypeptides were detected and the distribution among the salt eluates were 85, 83, 40 and 29 polypeptides, respectively. The 40 major polypeptides with regard to concentrations were also identified from gels stained with a protein-specific reagent. The polypeptides could be divided into two main groups according to pI values, namely, acidic polypeptides (total number, 174) and basic polypeptides (total number, 63). The ratio between acidic and basic polypeptides decreased with increasing salt concentrations in the eluates. The majority of the basic polypeptides had molecular weights in the range 10 000-30 000, whereas the acidic polypeptides had molecular weights from 10 000 to 165 000.     

Frog Virus 3 Replication: Analysis of Structural and Nonstructural Polypeptides in Infected BHK Cells by Acidic and Basic Two-Dimensional Gel Electrophoresis

Analysis of frog virus 3-infected BHK cells by two-dimensional, acidic and basic gel electrophoresis showed that at least 90 infected cell-specific polypeptides could be detected. These polypeptides represent between 70 and 85% of the coding capacity of the viral genome. The polypeptides were sequentially induced in at least three phases. The virus gradually suppressed host cell polypeptide synthesis during infection, although the synthesis of a few cell polypeptides may be “switched off” early in infection.     

The synthesis of acidic chromosomal proteins during the cell cycle of HeLa S-3 cells. II. The kinetics of residual protein synthesis and transport

The kinetics of acidic residual chromosomal protein synthesis and transport were studied throughout the cell cycle in HeLa S-3 cells synchronized by 2 mM thymidine block and selective detachment of mitotic cells. Pulse labeling the cells with leucine-³H for 2 min and then "chasing" the radioactive proteins for up to 3 hr showed that the amount of protein synthesized, transported, and retained in the acidic residual chromosomal protein fraction is greater immediately after mitosis and later in G₁ than in the S or G₂ phases of the cell cycle. During S, only 20–25% of the proteins synthesized and transported to the acidic residual chromosomal protein fraction are chased during the first 2 hr after pulse labeling, whereas up to 40% of the material entering the residual nuclear fraction in mitosis, G₁, and G₂ leaves during a 2 hr chase. Polyacrylamide gel electrophoretic profiles of these proteins, at various times after pulse labeling, reveal that the turnover of individual polypeptides within this fraction has kinetics of synthesis and turnover which are markedly different from one another and undergo stage-specific changes.     

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.     

Transformation-associated changes in nuclear-coded mitochondrial proteins in 3T3 cells and SV40-transformed 3T3 cells

Comparative two-dimensional gel electrophoretic studies were performed on mitochondrial proteins in nontransformed mouse 3T3 cells and in SV40-transformed 3T3 cells, SV-T2. Two polypeptides, of 58 and 40 kDa, were present in increased amounts in SV40-transformed cells. These polypeptides were demonstrated to be nuclear-coded mitochondrial proteins by their absence in mitochondrial preparations, when labeling was performed in the presence of a mitochondrial-specific inhibitor, Rhodamine 6G. Temperature-sensitive mutants for transformation were derived from 3T3 cells by transfection with cloned SV40 DNA containing the ts A58 mutation. Increased amounts of the 58 kDa protein were apparent in these cells at the permissive temperature (33 degrees C) compared to the restrictive temperature (39.5 degrees C).     

Growth effects of lithium chloride in BALB/c 3T3 fibroblasts and madin-darby canine kidney epithelial cells

The ability of LiCl to initiate DNA synthesis was studied in Madin-Darby canine kidney (MDCK) cells, and mouse BALB/c 3T3 fibroblasts. In a defined culture medium lacking serum, LiCl increased DNA synthesis in BALB/c 3T3 cells 100–200% over control values. Maximum DNA synthesis was observed with concentrations of LiCl between 10 and 25 mM and increases from 40–50% over control were observed with concentrations as low as 1 mM. Exposure of BALB/c 3T3 cultures to LiCl resulted in an increase in the percentage of cells initiating DNA synthesis, total DNA content and cell number. Lithium chloride, in combination with insulin or epidermal growth factor (EGF), had either an additive or synergistic effect upon the growth of BALB/c 3T3 fibroblasts. MDCK cells proved refractory to the growth actions of LiCl, although they responded to EGF and insulin with increased DNA synthesis. Lithium chloride appears to have a direct effect on cell proliferation in some but not all cell types.     

Two-dimensional polyacrylamide gel electrophoresis analysis of phosphorylated, membrane-localized ras p21 proteins

The ras p21 oncogene product migrates as a heterogeneous series of polypeptides as resolved by both one- and two-dimensional polyacrylamide gel electrophoresis (PAGE). We have prepared polyclonal rat serum antibody to ras p21 and used this as well as monoclonal antibodies to immunoprecipitate forms of p21 synthesized in vivo in transformed NIH3T3 cells. Two-dimensional PAGE of p21 resolved two distinct groups of polypeptides, one acidic (pI 4.8-5.3) which we call the "A" forms, and one less acidic or more basic (pI 6.5-7.0) which we call the "B" forms. It is the membrane-localized, B forms of v-ras-Ha p21 that are predominantly phosphorylated in vivo.     

Fibroblast Growth Factor stimulates anchorage independent growth in agar of BHK21/13 and SV3T3 cells

The effect of Fibroblast Growth Factor (FGF) on the growth in suspension in soft agar of secondary baby hamster kidney fibroblasts, the partially transformed cell line BHK21/13 and its virally transformed derivative SV28 was studied. Only BHK21/13 cells could be induced by FGF to form colonies in soft agar. Secondary baby hamster kidney fibroblasts did not grow in suspension even with FGF, whereas SV28 grew independently of the presence of FGF. Thus viral transformation of BHK21/13 was not a prerequisite for growth in agar if FGF was present. Epidermal Growth Factor (EGF) was ineffective at promoting growth in agar of secondary baby hamster kidney fibroblasts or BHK21/13 cells. Balb/c 3T3 behaves like the secondary baby hamster kidney and SV3T3 like BHK21/13. We conclude there are three degrees of transformation with different potentials for growth in agar.     

Sulfated mucopolysaccharides from normal and virus transformed rodent fibroblasts.

The sulfated mucopolysaccharide composition of normal and virus transformed Balb 3T3 and BHK21 cell lines is reported. It is shown that normal 3T3 cells contain mainly chondroitin sulfate B and heparitin sulfate. Relatively higher amounts of chondroitin sulface AC were observed in polyoma virus transformed 3T3 cells, besides an absolute increase of all the three sulfated mucopolysaccharides in the polyoma and SV 40 transformed cells. It is shown also that the three sulfated mucopolysaccharides are at least in part at the cell surface. Similar differences in sulfated mucopolysaccharide composition of normal and virus transformed BHK cell lines were also observed.     

Effect of Interferon on Some Aspects of Transformation by Polyoma Virus

WHEN BHK 21 hamster cells are infected with polyoma virus¹, there is no vegetative growth of virus, but stably transformed cells appear. These transformed cells are more easily transplanted than BHK 21 cells; they initiate their growth cycle in otherwise restrictive cultural conditions such as the absence of serum, high density and suspension; they grow with random orientation and have exposed on their surfaces receptor sites for certain glycoprotein agglutinins^2–5. The proportion of stably transformed cells is low, even after high doses of virus. But a much higher proportion (sometimes all cells) shows abortive transformation—changes characteristic of transformation, but which last only a few days. In suspension cultures, for example, most of the infected cells grow into small colonies of four to thirty-two cells⁶. In surface cultures deprived of serum DNA synthesis is initiated and the cells may then divide at least once⁷: they also temporarily lose the normal parallel orientation and develop the typical random appearance of transformed cells. Moreover, the polyoma nuclear T-antigen and also a surface antigen detected by immunofluorescence, appear temporarily in most polyoma infected BHK 21 cells⁸, while 3T3 cells exposed to SV40 virus show transient exposure of cell surface sites reacting with conconavalin A (ref. 9).     

Failure of an Influenza Virus to Initiate Infection in Enucleate BHK cells

Studies employing indirect immunofluorescent staining, acrylamide gel electrophoresis of [(35)S]methionine-labeled cellular polypeptides, and RNA-RNA hybridization of [(3)H]uridine-labeled cellular RNA, failed to detect evidence of fowl plague virus infection of BHK cells enucleated with cytochalasin B, although virus-specific polypeptide and RNA synthesis was detected in nucleate BHK cells. The enucleate cells permitted the synthesis of Newcastle disease and vaccinia virus structural proteins. We conclude that influenza virus fails to initiate macromolecular synthesis in enucleate BHK cells, and the role of the nucleus in influenza virus replication is discussed.     

Spatial differences in the presence of FOXP3+ and GranzymeB+ T cells between the intra- and extravascular compartments in renal allograft vasculopathy

Background

Allograft vasculopathy (AV) and native atherosclerosis (NA) share the presence of a T-cell mediated inflammatory response, but differ in overall plaque morphology and growth rate. We studied the distribution and frequency of regulatory- and cytotoxic T cells in the arterial intima lesions in both conditions.

Methodology/Principal Findings

The study is based on vessels of 15 explanted human renal allografts with AV and 10 carotid artery plaques obtained at surgery. Distribution and frequency of cytotoxic- and regulatory T cells, as identified by the expression of Granzyme B (GrB) and FOXP3 was established in NA and AV. Furthermore, we compared the distribution of these cells in AV with the perivascular, interstitial renal tissue using immunohistochemistry. The total number of T cells was much higher in AV than in NA lesions (711±135 and 37±8 CD3/mm² respectively, p<0.005, mean, ± SEM). Total numbers of FOXP3⁺ regulatory cells were also significantly increased in AV (36±10 and 0.9±0.3 FOXP3⁺/mm² p<0.05), but relative numbers, expressed as a percentage of the total number of CD3⁺ T cells ((FOXP3⁺/CD3⁺) ×100), were not significantly different (4.6%±0.9 and 2.7%±0.6). GrB⁺ cells were rare in NA, but significantly increased numbers of GrB⁺ cells were found in AV lesions (85±24 and 0.2±0.1 GrB⁺/mm², p<0.05). Perivascular tissues in the allografts showed a higher relative frequency of FOXP3⁺ cells than adjacent intimal lesions (14.0%±2.7 and 4.6%±0.9, respectively, p<0.05), but a lower frequency of GrB⁺ cytotoxic T cells (16.1%±2.7 and 22.6%±3.6, p<0.05).

Conclusions

Similar to NA, AV is characterized by a low frequency of intimal FOXP3⁺ regulatory T cells. Moreover, significant spatial differences exist in the distribution of functional T cell subsets between the intra- and extravascular micro-environments of the graft.     

Density dependent inhibition of cell growth in cultures of primary and established lines of cells

The effect of cell crowding on DNA synthesis (incorporation of ³HTdR and ³²PO₄) was studied by an improved method in monolayers of secondary cells and established cell lines, either normal or transformed by viruses or carcinogens. The method was based mainly on pulse labeling of cultures of cells a few hours after their seeding in equal numbers onto areas of different size in identical dishes, a condition which ensured equal physiological conditions and different degrees of crowding of cells. DNA synthesis was hardly inhibited in crowded monolayers of secondary chick, mouse and hamster embryo cells. The incorporation of radioactive thymidine and phosphate into DNA of cell lines such as BHK 21, 3T3/SV40 and L929 was strongly inhibited. An SV40-transformed line of hamster kidney cells (HKT7) synthetized DNA equally well in sparse as in crowded monolayers. In lines of human amnion (FL) and BHK 21 cells which were more extensively studied the degree of inhibition of DNA synthesis was inversely proportional to their density. Autoradiography after ³HTdR pulse-labeling indicated that the same proportion of cell nuclei were labeled in sparse and in crowded cultures. The extent of labeling (number of grains per nucleus) was lower in crowded cultures of those cells that also showed inhibition of incorporation of this label as measured by scintillation. The inhibition is thus expressed in retardation of DNA synthesis in cells in S phase rather than arresting it in a larger percentage of cells.