Sarcomas routinely produced from putatively nontumorigenic Balb/3T3 and C3H/10T1/2 cells by subcutaneous inoculation attached to plastic platelets

The Balb/3T3 and C3H/10T1/2 lines, noted for their marked postconfluence inhibition of proliferation and anchorage dependence, and frequently studied as nontumorigenic lines that are compared with tumorigenic sublines transformed with various agents, produced tumors within two to four months at low-cell dosage (3 × 10⁴ cells) when implanted subcutaneously attached to 1 × 5 × 10 mm polycarbonate platelets. Platelets alone did not produce tumors. The cultured Balb/3T3 tumor cells showed loss of both postconfluence inhibition of proliferation and anchorage dependence. Tumors arising form attached Balb/3T3 cells in (BALB/c × C57B1/6)F1 hybrids were shown to be transplantable to BALB/c but not to C57B1/6 mice, proving that the tumors were derived form Balb/3T3 and not from host cells. The tumors exhibited unique transplantation rejection antigens that did not cross-react with each other. Scanning electronmicroscopy of Balb/3T3 cells and derive tumor cells on Teflon

1 Teflon: Registered trademark of DuPont Plastics.

substrates (on which only the tumor cells and not the parent Balb/3T3 cells could grow) revealed that the two cell types were remarkably similar in appearance, except that the tumor cells were larger and showed many more microvilli that tended to concentrate over the nucleus. We conclude that Balb/3T3 cells and C3H/10T1/2 cells are preneoplastic and give rise to spontaneously transformed clones when implanted in vivo attached to a solid substrate.     

Contact-stimulated proliferation of cultured mouse epidermal cells by 3T3 feeder layers: Inhibition of proliferation by 12-O-tetradecanoylphorbol-13-acetate (TPA)

Mouse epidermal cells can be subcultured at 31°C onto an irradiated BALB/c 3T3 clone A31 feeder layer. A31 cells (supposedly derived from embryonic fibroblasts) were found to be specifically required for the optimal production of keratinizing epidermal colonies in secondary culture. This effect was not transmitted through the medium nor by the culture surface, since A31 cells plated on one end of a flask did not stimulate epidermal cell proliferation at the other end, even if the other end had previously held A31 cells. Epidermal cell contact with metabolizing A31 cells was probably necessary for the effect; fixed or freeze-thawed A31 cells were ineffective. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate, recently shown to interfere with contact-mediated transfer of label (metabolic cooperation) between Swiss 3T3 cells and cells of an established epidermal line in vitro, also blocked epidermal colony formation. The A31-epidermal cell interaction is apparently not a typical mesenchymal-epithelial interaction, since the basement membrane would prevent this contact in intact skin.     

Studies on morphological transformation of BALB/3T3-derived clones by murine leukemia virus.

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

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

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

Cell cultivation on microspheres coupled with histones

We have studied the application of histones for the modification of a cell-culture surface. Experiments were conducted on 293 cell lines (human embryonic kidney cells transformed by Ad5 adenovirus) and BALB/3T3 clone A31 (spontaneously transformed mouse embryonic fibroblasts). The cell’s interactions with various types of histons placed on a hydrophobic glass surface or 1.0 μm dextran microspheres was assessed. It was found that all histones studied exhibited adhesive properties but their complexes, such as the total and core histones, were the most adhesive and had improved cell morphology. Cross-linked conjugates of histone complexes immobilized on microshperes facilitated cell network formation by cellular interactions and cell contacts with several microspheres. For the 293 line, unlike BALB/3T3 clone, the A31 cells significantly increased their proliferative activity on microspheres coated with cross-linked conjugates of histone complexes. The study showed that the substrate may be applied for 3D pore matrices designed for tissue-like cell structures in vitro.     

Malignant transformation of mouse BALB/3T3 cells by polyoma middle T antigen requires epidermal growth factor receptor expression.

The mouse cell line MO-5, which is defective in receptor-binding activity of epidermal growth factor (EGF), is very poorly transformed by polyoma middle T antigen or v-src gene, but activated c-H-ras and v-mos gene can induce the transformation (M. Ono, M. Yakushinji, K. Segawa, and M. Kuwano, Mol. Cell. Biol., 8: 4190-4196, 1988). We established clones of MO-5 expressing a functional EGF receptor (EGF-R) after introduction of the human EGF-R complementary DNA into MO-5 (MNER23 and MNER31), and we also established a clone (BNER4) expressing human EGF-R from the parental cell line, BALB/3T3. MNER23, MNER31, and BNER4 expressed EGF-R activity at about 2- to 6-fold higher levels than did control BALB/3T3 cells. A marked increase in DNA synthesis in response to EGF was observed in these BNER4, MNER23, and MNER31 cell lines compared to BALB/3T3 cells; however, there was little if any increase in DNA synthesis of MO-5 in the presence of EGF. Introduction of the polyoma middle T antigen gene into BALB/3T3, BNER4, MNER23, and MNER31 resulted in the appearance of transformation foci, but MO-5 again showed little response. We purified clones B4-mT-2, M23-mT-1, M23-mT-2, M23-mT-3, and M31-mT-13 from transformation foci of BNER4, MNER23, and MNER31 cells, which were respectively transfected with the middle T antigen. All of the middle T antigen-positive transfectants demonstrated abilities to form both colonies in soft agar and tumors in nude mice. The presence of EGF-R appears to be indispensable for malignant transformation by polyoma middle T antigen.     

Long-term maintenance of “cloned” human PLT cells in TCGF with LCL cells as a feeder layer

The long-term maintenance of T cells “cloned” by limiting dilution in TCGF was enhanced by the use of irradiated autologous lymphoblastoid cell line (LCL) cells as well as irradiated LCL cells of the individual to which the T cells were originally primed. It was possible to obtain more than 1 × 10¹² cells from a “clone” seeded at one cell per well. Some of the clones tested express primed LD-typing activity.     

Effect of embryonic mouse cells BALB/c-3T3 on the proliferation of the human mammary cancer cell line T-47D

In the present study, we explore the effect of the cellular extracts and culture medium of the embryonic mouse cell line BALB/c-3T3 (clone A31) on the proliferation and DNA content of the human T-47D breast cancer cell line. These effects were also studied in the presence of the potent anti-estrogen ICI 164,384. All experiments were prepared in MEM medium containing 5% fetal calf serum treated with dextran charcoal, as well as the homogenization of the BALB/c-3T3 cells to obtain the cellular extract. Aliquots of cellular extracts (2%) corresponding to 2 × 10⁶ cells, or culture medium (16%), are incubated with the T-47D cells. After 9 days of culture, cellular extracts and culture medium provoke an intense proliferative effect corresponding respectively to 2 and 5 times the control value of T-47D cells. These effects on cell proliferation are correlated with DNA content. Although the anti-estrogen ICI 164,384 (5 × 10⁻⁸ M) alone decreases the proliferation of T-47D cells by half, the presence of the culture medium from the BALB/c-3T3 cells abolishes this effect and, on the contrary, increases the cell proliferation 4-fold.

It is concluded that mouse embryonic cells (BALB/c-3T3) contain factor(s) which stimulate very intensively the proliferation of hormone-dependent T-47D mammary cancer cells. This factor(s) is present in both the cell and the culture medium and can antagonize the anti-proliferative effect of the anti-estrogen ICI 164,384.     

Online monitoring of BALB/3T3 metabolism and adhesion with multiparametric chip-based system

A multiparametric chip-based system was employed to measure cell adhesion, metabolism, and response to metal compounds previously classified as cytotoxic in immortalized mouse fibroblasts (BALB/3T3 cell line). The system measures in parallel, online, and in label-free conditions the extracellular acidification rates (with pH-sensitive field effect transistors [ISFETs]), the cellular oxygen consumption (with amperometric electrode structures [Clark-type sensors]), and cell adhesion (with impedimetric interdigitated electrode structures [IDESs]). The experimental protocol was optimized to monitor metabolism and adhesion of the BALB/3T3 cell line. A total of 70,000 cells and a bicarbonate buffer-free running low-glucose Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal clone serum III and 1mM Hepes were selected to maintain cells in good conditions on the chip during the measurements performed under perfusion conditions. Cells were exposed to sodium arsenite, cadmium chloride, and cis-platinum at concentrations ranging from 1 to 100 microM. The kinetics of cell response to these compounds was analyzed and suggests that the Clark-type sensors can be more sensitive than IDESs and ISFETs in detecting the presence of high chemical concentration when short exposure times (i.e., 2h) are considered. The cytotoxicity data obtained from the online measurements of acidification, respiration, and adhesion at 24h compare well, in terms of half-inhibition concentration values (IC(50)), with the ones obtained using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test and colony-forming efficiency (CFE) assay. The results show a good sensitivity of the system combined with the advantages of the online and label-free detection methods that allow following cell status before, during, and after the treatment in the same experiment.     

Analysis of autocrine growth regulators produced by chondrogenic fibroblasts of chick embryo sclera in protein-free primary cultures

Abstract. In the chick embryo there is a population of chondrogenic fibroblasts known as scleral fibroblasts. Scleral fibroblasts in primary culture secrete multiple autocrine growth-promoting factors, scleral autocrine factors (SAFs), into protein-free medium (Watanabe et al . 1989). One such factor, SAF-IIa, which is heat-labile and binds to heparin, shows strong DNA synthesis-promoting activity on the mouse fibroblast cell line, BALB/c 3T3 A31 cells and has a molecular weight of c . 16 kDa by gel filtration. These data suggest that SAF-IIa is related to growth factors of the FGF family. However, the effects of heparin augmentation on the growth-promoting activity suggest that SAF-IIa is not identical to aFGF or bFGF, when assayed on scleral fibroblasts and also on BALB/c 3T3 A31 cells. The other heat-labile autocrine growth-promoting factor, SAF-IIb, shows weak binding to heparin and no growth-promoting activity for BALB/c 3T3 A31 cells. The heat-resistant growth factor, SAF-I, is effective in enhancing the proliferation of BALB/c 3T3 cells, and its activity is increased by heat treatment. Whole-embryo fibroblasts, which show low autocrine growth in protein-free medium, produce mainly SAF-IIa-like growth-promoting activity and do not produce SAF-I. This indicates that the strong proliferative activity of scleral fibroblasts in vitro can be attributed to the production of a strong and stable autocrine factor, SAF-I, in the growing phase (Watanabe et al . 1989) and this is a specialized property of the chondrogenic cells of the sclera.     

Neoplasms produced from C3H/10T 1/2 cells attached to plastic plates; saturation density, anchorage dependence and serum requirement of in vitro lines correlated with growth aggressiveness in vivo

The C3H/10T 1/2 embryo cell line, which is nontumorigenic when inoculated subcutaneously in saline suspension, produces tumors when implanted subcutaneously attached to 1 X 5 X 10 mm plastic plates. Under these in vivo conditions there is direct selection for "spontaneous" transformants that have undergone the specific cellular alterations required for neoplastic behavior. This is in contrast to the conventional situation where transformants are obtained in vitro and are only secondarily tested in vivo for neoplastic behavior. Early passages of cell lines from four different C3H/10T 1/2 tumors explanted back in culture were quantitatively examined for tumorigenicity and for alteration in the properties of density inhibition, anchorage dependence, serum requirement, and plasminogen activator production. A fairly consistent quantitative relationship was found between the degree of growth aggressiveness in vivo and the degree of expression of these phenotypic markers of the transformed state in vitro during early passages of the cell lines after tumor explantation.     

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.     

Human recombinant insulin-like growth factor I. II. Binding characterization and radioreceptor assay development using BALB/c 3T3 mouse embryo fibroblasts

Summary The binding of human recombinant insulin-like growth factor I (IGF-I) to BALB/c 3T3 mouse embryo fibroblasts has been characterized, resulting in the development of a radioreceptor assay. Binding of radioiodinated IGF-I (¹²⁵I-IGF-I) to washed monolayers of BALB/c 3T3 cells was specific, time dependent, and stable, being maximal after a 10-h incubation at 15°C with no loss of bound ligand or cells through 25 h. Scatchard analysis identified a class of high affinity binding sites with K _d =59.6 pM and an estimated 1.57×10⁵ receptors/cell. Half-maximal displacement of bound¹²⁵I-IGF-I occurred with 15 to 20 ng/ml unlabeled IGF-I competitor. Insulin-like growth factor II and insulin were far less effective competitors, providing halfmaximal displacement at concentrations of 130 to 170 ng/ml and 2 to 3 μg/ml, respectively. Epidermal growth factor, transforming growth factor type α, and acidic and basic fibroblast growth factors did not compete for¹²⁵I-IGF-I binding at 1 μg/ml. Cells fixed with glutaraldehyde before ligand binding did remain attached to culture dishes more tightly; however such pretreatment destroyed approximately 70% of ligand binding. Crosslinking data indicated that¹²⁵I-IGF-I binds specifically to a 330-kDalton receptor as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. This receptor dissociated into 130-kDalton subunits when analyzed in the presence of dithiothreitol. This work was supported by a contract from IMCERA Bioproducts, Inc.     

Control by cell interaction of phosphate uptake in 3T3 cells.

Phosphate uptake by monolayers of 3T3 cell decreases when the cultures enter the stationary phase, even when incubated in fresh medium containing 10% serum. However, SV 3T3 cultures retain a high rate of phosphate uptake when the cells reach saturation densities.We have observed that 3T3 cells grown to stationary phase in monolayers and then trypsinized and incubated in suspension, display an increase in phosphate uptake when the cell concentration is decreased from 10⁶ cells/ml to 10⁵ cells/ml. Where the cell concentration is further reduced from 10⁵ cells/ml to 2.5 × 10⁴ cells/ml there is no further increase in the rate of phosphate uptake. We observed, on the contrary, a small decrease.The “concentration effect” (the decrease of phosphate uptake when the cell concentration increases from 10⁵ to 10⁶ cells/ml) is larger when cells originate from a culture in stationary phase than when they originate from a culture in log phase.The “concentration effect” may be observed 10 min after cell incubation but is larger after a lag time of 40 min incubation.Differences in the “concentration effect” may be noted between 3T3 and SV 3T3 cells. In SV 3T3 cells no significant variations of phosphate uptake were observed when the cell concentration was changed. Thus, differences between phosphate uptake in 3T3 and SV 3T3 cells are large when cells are incubated at high concentrations or at high densities and small when they are incubated at low concentrations or at low densities.The “concentration effect” in 3T3 cells supports the assumption that interactions between cells cause the decrease of phosphate metabolism in dense culture. Diffusion of an inhibitor into the medium remains the more plausible explanation of the data.     

Generation of megakaryocytes and platelets from preadipocyte cell line 3T3-L1, but not the parent cell line 3T3, in vitro

Platelets are produced from megakaryocytes (MKs), although the pathway leading from stem cells to MK lineages are not yet fully understood. Recently, we reported to obtain abundant MKs and platelets from human subcutaneous adipose tissues. Adipose tissues contain various cell types, most of which are lineage cells from mesenchymal or adipocyte-derived stem cells, distinct from hematopoietic cells. To identify the cells responsible for the differentiation MK lineages in adipose tissues, this study examined whether the preadipocyte cell line 3T3-L1 and fibroblast cell line 3T3 differentiated into MK lineages in vitro. Cells were cultured in megakaryocyte lineage induction medium. By day 4, most of 3T3 cell-derived cells leaded to cell death. In contrast, 3T3-L1-derived cells on days 8 showed to have typical characterizations of MK lineages in analyses for specific marker, DNA ploidy, transmission electro micrograph. 3T3-L1-derived platelet-sized cells on day 12 could be stimulated by ADP and PAR4-activating peptide. This study clearly shows in vitro differentiation from 3T3-L1 cells, not from 3T3 cells, into MK lineages.     

Coordinate regulation of the levels of type III and type I collagen mRNA in most but not all mouse fibroblasts

A mouse genomic clone was isolated by cross-hybridization with a DNA fragment which codes for the NH2-propeptide of chick alpha1(III) collagen. The region of cross-hybridization within the mouse clone was localized, its sequence determined, and an exon coding for the NH2-propeptide of mouse alpha1(III) collagen was identified. This DNA fragment hybridizes to an RNA species of approximately 5300 nucleotides, slightly larger than the major alpha2(I) collagen RNA species. The mouse type III collagen probe was used to examine the effect of transformation on alpha1(III) collagen RNA levels in mouse fibroblasts. The levels of type III and type I collagen mRNA levels were compared in control and sarcoma virus-transformed murine cell lines, as well as in NIH 3T3 cells transformed by members of the human ras oncogenes. The levels of type III RNA decreased about 10-15-fold in Moloney sarcoma virus-transformed cells and in a cell line transformed with a v-mos-containing plasmid, but showed only a 50% decrease in a Kirsten murine sarcoma virus-transformed BALB 3T3 cell line, and increased 4-fold in a Rous sarcoma virus (RSV)-transformed BALB 3T3 cell line. In contrast, the levels of alpha2(I) collagen mRNA are 8- to 10-fold lower in all these cell lines when compared to untransformed cells. NIH 3T3 cells transformed with two human ras oncogenes showed decreased levels of alpha2(I) and alpha1(III) mRNAs. In contrast to the RSV-transformed mouse cell line, RSV-transformed chick embryo fibroblasts contained much smaller amounts of type III RNA than control chick embryo fibroblasts. We conclude that the levels of alpha1(III) and alpha2(I) collagen mRNA are often but not necessarily coordinately regulated by transformation in mouse cells.     

Trypanosoma brucei: influence of host strain and parasite antigenic type on infections in mice

Inbred mice were infected with cloned populations of Trypanosoma brucei brucei Lister S42 under carefully controlled conditions. The course of infection was found to depend both on host strain and the antigenic type of the infecting organisms. The two antigenic types used, “NIM2” and “NIM6” had differing virulence for (CBA/H × C57BL/6)F₁ mice, and when mice were infected with parasites of one clone, trypanosomes of the other type frequently appeared after the initial population had been eliminated. Different mouse strains had varying susceptibility to clone NIM6. In most cases there was an inverse relation between the survival time and the parasite load during the first peak of parasitemia. The differences in resistance to T. brucei were unrelated to H-2 haplotype: C57BL/6 and (CBA/H × C57BL/6)F₁ were most resistant, CBA/H, BALB/c and DBA/2 less so, and C3H/He most susceptible.     

3T3-L1 adipocytes promote the growth of mammary epithelium

Murine mammary epithelium grows in association with predominantly adipocyte stroma in vivo. To investigate potential growth-promoting effects of adipocytes on mammary epithelium, we developed a co-culture system of mammary epithelium and adipocytes by taking advantage of the 3T3-L1 cell line. These cells undergo adipocyte differentiation when the culture reaches confluence and growth ceases. Mid-pregnant murine mammary epithelium was plated on lethally irradiated feeder layers of 3T3-L1 adipocytes, undifferentiated 3T3-L1 cells, 3T3-C2 fibroblasts (a subclone of 3T3 cells that does not undergo adipocyte differentiation), or tissue culture plastic. Mammary epithelial colony size on adipocyte feeder layers was 2-fold larger than colonies on 3T3-C2 cells and 4-fold larger than colonies on tissue culture plastic. Measurement of tritiated thymidine [3H]TdR incorporation and labelling index in mammary cells was significantly higher on adipocytes than on other feeder layers or plastic. There was a 6-fold increase in mammary cell number after 5 days in culture when mammary epithelium was plated on substrate-attached material ('extracellular matrix') derived from 3T3-L1 cells and a 4-fold increase in cell number when plated on plastic in conditioned medium derived from 3T3-L1 adipocytes compared with growth on plastic in unconditioned medium. We conclude that interaction of mammary epithelium with adipocytes results in a marked increase in proliferation of mammary epithelium and that extracellular components may mediate this effect.     

S100A6 (calcyclin) deficiency induces senescence‐like changes in cell cycle,morphology and functional characteristics of mouse NIH 3T3 fibroblasts

S100A6 (calcyclin) is a calcium binding protein with two EF‐hand structures expressed mostly in fibroblasts and epithelial cells. We have established a NIH 3T3 fibroblast cell line stably transfected with siRNA against S100A6 to examine the effect of S100A6 deficiency on non‐transformed cell physiology. We found that NIH 3T3 fibroblasts with decreased level of S100A6 manifested altered cell morphology and proliferated at a much slower pace than the control cells. Cell cycle analysis showed that a large population of these cells lost the ability to respond to serum and persisted in the G0/G1 phase. Furthermore, fibroblasts with diminished S100A6 level exhibited morphological changes and biochemical features of cellular senescence as revealed by β‐galactosidase and gelatinase assays. Also, S100A6 deficiency induced changes in the actin cytoskeleton and had a profound impact on cell adhesion and migration. Thus, we have shown that the S100A6 protein is involved in multiple aspects of fibroblast physiology and that its presence ensures normal fibroblast proliferation and function. J. Cell. Biochem. 109: 576–584, 2010. © 2009 Wiley‐Liss, Inc.