Stimulation of cell growth and proliferation in NIH-3T3 cells by onion and garlic oils

Onion and garlic oil were seen to shorten the cell-doubling time and stimulate the growth and proliferation of NIHY-3T3 cells. FNollowing treatment with either onion or garlic oil, an increase in the growth rate and almost a 2-fold increase in cell number over the control was observed within a 24-hour period. Phorbol myristate acetate when given simultaneously with either oil appeared to nullify both effects. FNollowing exposure to low doses (< 10 µm/ml) of either oil, an increase in cell survival, not seen with the oil control tricaprylin, was observed following a 5-day exposure period. At higher concentrations cell survival decreased proportionately in all cases. The appearance of multinucleated cells, which increased with dose and time, was also observed following treatment with both garlic and onion oil.Abbreviations EMEMM Eagles Minimal Essential Medium - MCN mean cell number - PMA phorbol myristateacetate - RCE relative cloning efficiency - SEM standard error of the mean     

Enhanced phospholipase C stimulation and transformation in NIH-3T3 cells expressing Q209LGq-alpha-subunits.

NIH-3T3 cells were transfected with cDNA encoding the native alpha-subunit of the G protein Gq(alpha q) or a mutant (Q209L) form of alpha q. Cells expressing Q209L-alpha q showed greatly enhanced basal phospholipase C activity. Stimulation of phospholipase C activity by prostaglandin F2 alpha or fetal calf serum was increased up to 10-fold in Q209L-alpha q-transfected cells. Continuous expression of Q209L-alpha q or overexpression of alpha q in NIH-3T3 cells resulted in formation of foci after 3 weeks. The number of foci was proportional to the number of transfected cells and was greater in cells expressing the Q209L-alpha q than in cells that overexpressed the wild type alpha q. Q209L-alpha q-transfected NIH-3T3 cells also formed colonies in soft agar indicating their capacity to grow in an anchorage-independent manner. Expression of Q209L-alpha q in Rat-1 cells resulted in enhanced basal and fetal calf serum-stimulated phospholipase C activity, but these cells were not transformed as assessed by either the focus formation or the soft agar colony formation assays. These results indicate that expression of continuously activated Gq-alpha can result in transformation in a cell type-specific manner.     

Modulation of protein synthesis in rabbit inner cell mass-derived cells by FGF-2

Gastrulation is a critical step in vertebrate development, that depends on synergistic effects of several signalling molecules, including fibroblast growth factor-2 (FGF-2). To follow this phenomenon in vitro we isolated rabbit inner cell masses (ICMs) at embryonic day 4 and we exposed ICM-derived cells to FGF-2. Then, we analysed the quantitative differences in rates of protein synthesis from day 3 to day 5 of culture by two-dimensional (2D) gel electrophoresis. Here we show that both up- and down-regulation of protein synthesis took place in ICM-derived cells upon their exposure to FGF-2. The effect of FGF-2 was most pronounced at day 4 of culture, when the changes were very much in favour of a set of down-regulated proteins. To test the significance of this period of time for FGF-2-mediated regulation of protein synthesis, cells were grown without FGF-2 and then they were pulse-treated with FGF-2 at the end of day 4. When compared to the continuous culture with FGF-2, the FGF-2 pulse resulted in a quite indistinguishable pattern of up- and down-regulated proteins. Thus, the readiness of ICM-derived cells to accept and respond to the FGF-2 signals may be of limited duration.     

Retinoids induce tissue transglutaminase in NIH-3T3 cells.

We report that all-trans and 13-cis-retinoic acid as well as the synthetic compound CH-55 enhance tissue transglutaminase activity as they increase NIH-3T3 cell adhesiveness. The 4-hydroxyphenylretinamide (4-HPR) with low activity in inducing attachment, lectin binding and growth inhibition also fails to induce transglutaminase. Thyroxine (Thy), a compound with a response element common to RA, is inactive. The tumor promoter 12-tetradecanoyl-phorbol-13-acetate (TPA), which increases adhesiveness with different kinetics than RA, failed to enhance tranglutaminase. We conclude that retinoids with biological activity in inducing adhesion, inhibition of growth and increase of lectin binding, are also active in inducing transglutaminase activity.     

Soluble form of the ST2 gene product exhibits growth promoting activity in NIH-3T3 cells

The ST2 gene is induced in murine fibroblast cells at the start of cell proliferation. Although IL-33 has been identified as a ligand for one of the two major gene products of ST2 – namely, the transmembrane receptor form ST2L – prompting immunological research on inflammation, the roles of the ST2 gene products in cell proliferation remain to be elucidated.Using a cell proliferation assay system with NIH-3T3 cells, a normal murine fibroblast cell line, we found that treatment with recombinant ST2 caused an acceleration of cell proliferation, suggesting that ST2 acts in an autocrine/paracrine fashion. Strikingly, shRNA-induced knockdown of both ST2 gene products, ST2 and ST2L, reduced cell proliferation. This effect was effectively canceled by the expression of shRNA-resistant ST2, but not shRNA-resistant ST2L.The novel enhancement of cell proliferation by ST2 appears to involve positive feedback. Since the ST2 level is increased in various diseases involving inflammation, future investigations into the role of ST2 gene products in relation to various diseases, including malignancies, may be warranted.     

Expression of a human insulin-like growth factor II cDNA in NIH-3T3 cells

Recombinant human insulin-like growth factor II (IGF-II) was produced in NIH-3T3 cells transfected with a plasmid containing a construct encoding the signal peptide and the sequence of mature human IGF II. Successfully transfected clones secreted correctly processed recombinant human IGF II at rates of about 100ng per 24 hours per 10(6) cells. The biological activity of the purified recombinant human IGF II exhibited similar potencies as standard human IGF II isolated from serum in radio-receptorassays as well as in thymidine incorporation assays.     

Geranylgeranylated, but not farnesylated, RhoB suppresses Ras transformation of NIH-3T3 cells

RhoB is a low molecular weight GTPase that is both farnesylated (RhoB-F) and geranylgeranylated (RhoB-GG) in cells. Based on data from rodent cell models, it has been suggested that RhoB displays differential effects on cell transformation, according to the nature of its prenylation. To test directly this hypothesis, we generated GTPase-deficient RhoB mutants that are exclusively either farnesylated or geranylgeranylated. We show that in Ras-transformed murine NIH-3T3 cells, RhoB-F enhances, whereas RhoB-GG and RhoB (F/GG) suppresses anchorage-dependent and -independent cell growth as well as tumor growth in nude mice. We then demonstrate that Ras constitutive activation of the tumor survival pathways Akt and NF-kappa B are blocked by RhoB-GG, but not by RhoB-F, providing further support for the opposing role of RhoB-F and RhoB-GG in Ras malignant transformation in NIH-3T3 cells. In addition, both RhoB (F/GG) and RhoB-GG induce apoptosis in Ras-transformed NIH-3T3 cells whereas RhoB-F has no effect. Our data demonstrate that RhoB-F and RhoB-GG which differ only by a 5-carbon isoprene behave differently in rodent cells highlighting the important role of prenyl groups in protein function and emphasize the potency of RhoB to regulate negatively the oncogenic signal.     

Proliferation- and cell cycle-dependent differences in expression of the 170 kilodalton and 180 kilodalton forms of topoisomerase II in NIH-3T3 cells.

The cellular content of 170kD and 180kD topoisomerase II was studied as a function of the proliferation state and cell cycle position in NIH-3T3 cells. When the cells were synchronized by serum starvation and then stimulated to enter the cell cycle by addition of fresh growth medium, the amount of 170kD topoisomerase II present was undetectable until the cells reached late S phase, peaked in G2-M phase cells, and decreased as the cells completed mitosis. The amount of 180kD topoisomerase II was constant once the cells entered the cell cycle. When exponentially growing cells were induced to enter G0 by serum starvation, the amount of 170kD topoisomerase II decreased in parallel with the loss of cells from the S and G2-M phases of the cell cycle and was undetectable once all of the cells reached G0. In contrast, the 180kD enzyme was still present after all of the cells had entered G0. The tightness of association of the two enzymes with chromatin was measured by determining the concentration of salt required to extract them from isolated nuclei. The 180kD enzyme required a higher concentration of NaCl for extraction than did the 170kD enzyme. The different patterns of expression of the two forms of topoisomerase II suggest that they perform different functions in cells.     

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.     

Differential effects of novel tumour-derived p53 mutations on the transformation of NIH-3T3 cells

The p53 tumour suppressor gene is frequently mutated in human tumours and different tumour-derived mutations have varying effects on cells. The effect of a novel tumour-derived p53 mutation and two recently described mutations from South African breast cancer patients on the growth rate, colony formation, cell cycle arrest after irradiation and response to chemotherapeutic drugs was investigated. None of the p53 mutations had any significant effect on the inherent growth rate of the cells; however, contact inhibition of growth in two of the mutants was lost. These same two mutants formed colonies in soft agar, whereas the third mutant did not. All three of the mutants failed to show a G(1) cell cycle arrest after exposure to 7 Gy of [(60)Co] radiation, albeit to different degrees. Cells expressing the p53 mutants were either more sensitive to cisplatin and melphalan or more resistant than the untransfected cells, depending on the mutation. However, there was no difference in response to daunorubicin treatment. These results demonstrate that different p53 mutations exert varying biological effects on normal cells, with some altering checkpoint activation more effectively than others. The data also suggest that the nature of the p53 mutation influences the sensitivity to cytostatic drugs.     

Insulin-like growth factor II overexpression does not affect sorting of lysosomal enzymes in NIH-3T3 cells.

The sorting of newly synthesized mannose 6-phosphate (M6P)-containing proteins and of the major excreted protein (MEP), a lysosomal thiol proteinase, was studied in NIH-3T3 cells transfected with the cDNA of human insulin-like growth factor II (IGF II) or with the vector alone. Extracts from media and cells labelled with [35S] methionine were used for chromatography on a M6P/IGF II receptor affinity matrix or for immunoprecipitation to assess the distribution of newly synthesized M6P-containing proteins and MEP, respectively. The results indicate that the overexpression of IGF II did not affect the synthesis and the sorting of M6P-containing proteins and of MEP. The binding and uptake of the lysosomal enzyme arylsulfatase A were not affected in IGF II overexpressing cells.     

Phospholipase D activation by the mitogens platelet-derived growth factor and 12-O-tetradecanoylphorbol 13-acetate in NIH-3T3 cells

The effect of mitogens on phospholipase D activity was investigated in NIH-3T3 fibroblasts by measuring the accumulation of phosphatidylpropanol, produced by phospholipase D phosphatidyl transferase activity when 1-propanol acts as the phosphatidyl group acceptor. Platelet-derived growth factor (PDGF) and 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulated phosphatidylpropanol production by the cells. The dose-response relationships for activation of phospholipase D and stimulation of thymidine incorporation by PDGF and TPA were comparable. The possibility that activation of phospholipase D is utilized by mitogens as a trans-membrane pathway for signalling cell growth is discussed.     

Malignant transformation of NIH-3T3 and CV-1 cells by a helical mycoplasma,Spiroplasma mirum,strain SMCA

Summary A helical mycoplasma,Spiroplasma mirum strain SMCA, produced malignant transformation in mouse NIH 3T3 cells and monkey kidney CV-1 cells. The transformed cells exhibited morphological changes consistent with the transformed phenotype, grew in soft agar and produced tumors in athymic and BALB/c mice. Transmission electron microscopy revealed structures morphologically similar to mycoplasmas present in the cytoplasm of transformed but not untransformed 3T cells. The time of inoculation ofS. mirum SMCA to 3T3 cells and the passage level of 3T3 cells affected transformation. Editor's statement This paper describes the possible role of a mycoplasma organism, which induces cataracts and brain pathology similar to Creutzfeld Jacob and other degenerative diseases, in malignant transformation of mammalian cells. In addition to this surprising and novel finding is the observation that the mycoplasma resides in an intracellular position. These findings may have important implications for understanding malignant transformation and the nature of the diseases produced by this organism.     

Modulation of FGF-2 binding to chondrocytes from the developing growth plate by perlecan.

FGF-2 is a regulator of chondrocyte proliferation in the developing growth plate and has been shown to bind to perlecan, a heparan sulfate proteoglycan. We evaluated the effect of perlecan isolated from the growth plate on the binding of FGF-2 to its low and high affinity receptors on resting and proliferating chondrocytes. Chondrocytes were isolated by pronase/collagenase digestion of 1 mm thick slices from the resting and proliferating zones of fetal bovine ribs and were plated in serum-free DMEM. Chondrocytes maintained their zone-specific level of DNA and matrix synthesis over a two-day culture period. The collagen, aggrecan, and perlecan components of the matrix produced were associated with the cell layer and were secreted into the medium. Most of the perlecan made by the chondrocytes was secreted into the medium. Western blots showed medium perlecan to contain two high molecular weight core proteins and overlay assays showed only the large core protein bound FGF-2. Cell layer perlecan contained only the smaller core protein. Immunoprecipitation assays of media showed that the medium perlecan bound (125)I-FGF-2, that the bound FGF-2 was eluted from perlecan by 2 M NaCl at pH 7.4, and that this binding was eliminated by prior digestion with heparatinase. This indicates that the perlecan secreted into the medium is a low affinity receptor for FGF-2. (125)I-FGF-2 also bound to the chondrocytes in cell culture. Competition studies showed exogenous FGF-2 reduced (125)I-FGF-2 binding to high affinity receptor but not the low affinity receptor in the cell layer. Exogenous perlecan, however, reduced (125)I-FGF-2 binding to both the low and the high affinity receptors in the cell layer by approximately 60%. The results suggest that perlecan made by growth plate chondrocytes is a low affinity receptor for FGF-2 and acts to sequester FGF-2 away from the high affinity receptor.