sepharose and Cell-Transformation--Neoplastic

Both oxidative/nitrosative stress and alterations in DNA methylation are observed during carcinogenesis of different tumor types, but no clear correlation between these events has been demonstrated until now. Melanoma cell lines were previously established after submitting the nontumorigenicmelanocyte lineage, melan-a, to cycles of anchorage blockade. In this work, increased intracellular oxidative species and nitric oxide levels, as well as alterations in the DNA methylation, were observed after melan-a detachment, which were also associated with a decrease in intracellular homocysteine (Hcy), an element in the methionine (universal methyl donor) cycle. This alteration was accompanied by increase in glutathione (GSH) levels and methylated DNA content. Furthermore, a significant increase in dnmt1 and 3b expression was identified along melan-a anchorage blockade. L(G)-Nitro-L-arginine methyl esther (L-NAME), known as a nitric oxide synthase (NOS) inhibitor, and N-acetyl-L-cysteine (NAC) prevented the increase in global DNA methylation, as well as the increase in dnmt1 and 3b expression, observed during melan-a detachment. Interestingly, both L-NAME and NAC did not inhibit nitric oxide (NO) production in these cells, but abrogated superoxide anion production during anchorage blockade. In conclusion, oxidative stress observed during melanocyte anchorage blockade seems to modulate DNA methylation levels and may directly contribute to the acquisition of an anoikis-resistant phenotype through an epigenetic mechanism.

Topics: Acetylcysteine; Animals; Anoikis; Cell Adhesion; Cell Culture Techniques; Cell Transformation, Neoplastic; Cells, Cultured; Cysteine; DNA Methylation; Gene Expression Regulation; Glutathione; Homocysteine; Lipid Peroxidation; Melanocytes; Mice; NG-Nitroarginine Methyl Ester; Nitric Oxide; Oxidative Stress; Sepharose; Superoxides; Trypsin

Tight junctions (TJs) regulate epithelial cell polarity and paracellular permeability. Loss of functional TJs is commonly associated with epithelial cell-derived cancers. Raf1-mediated transformation of rat salivary gland epithelial cells (Pa4-Raf1) induces transcriptional downregulation of the TJ protein occludin and forced re-expression of occludin rescues polarized phenotype of epithelial cells. In the present study, we used this model to examine how specific structural modifications in the occludin protein affect its function in vitro and influence tumor growth in vivo. Our results revealed that neither the C-terminal nor the N-terminal half of occludin alone were sufficient to rescue cells from transformation by Raf1. However, forced expression of an occludin mutant lacking the first extracellular loop was sufficient to rescue cells from Raf1-mediated transformation. Interestingly, forced expression of an occludin mutant lacking the second extracellular loop did not rescue the epithelial phenotype in vitro nor did it prevent tumor growth in vivo. These results demonstrate that the TJ protein occludin has a potent inhibitory effect on the Raf1-mediated tumorigenesis, and the second extracellular loop of occludin appears to be critical for this function.

Topics: Animals; Cell Adhesion; Cell Line; Cell Line, Transformed; Cell Proliferation; Cell Shape; Cell Transformation, Neoplastic; Epithelial Cells; Humans; Membrane Proteins; Mice; Mice, Nude; Occludin; Phenotype; Proto-Oncogene Proteins c-raf; Rats; Sepharose; Tight Junctions; Tumor Suppressor Proteins

Stem cell factor (SCF) delays differentiation and enhances the expansion of erythroid progenitors. Previously, we performed expression-profiling experiments to link signaling pathways to target genes using polysome-bound mRNA. SCF-induced phosphoinositide-3-kinase (PI3K) appeared to control polysome recruitment of specific mRNAs associated with neoplastic transformation. To evaluate the role of mRNA translation in the regulation of expansion versus differentiation of erythroid progenitors, we examined the function of the eukaryote initiation factor 4E (eIF4E) in these cells. SCF induced a rapid and complete phosphorylation of eIF4E-binding protein (4E-BP). Overexpression of eIF4E did not induce factor-independent growth but specifically impaired differentiation into mature erythrocytes. Overexpression of eIF4E rendered polysome recruitment of mRNAs with structured 5' untranslated regions largely independent of growth factor and resistant to the PI3K inhibitor LY294002. In addition, overexpression of eIF4E rendered progenitors insensitive to the differentiation-inducing effect of LY294002, indicating that control of mRNA translation is a major pathway downstream of PI3K in the regulation of progenitor expansion.

Topics: 5' Untranslated Regions; Animals; Blotting, Western; Cell Differentiation; Cell Line; Cell Transformation, Neoplastic; Chromones; DNA Primers; DNA, Complementary; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Erythrocytes; Eukaryotic Initiation Factor-4E; Flow Cytometry; Genetic Vectors; Guanosine Triphosphate; Hemoglobins; Immunoprecipitation; Mice; Microscopy, Fluorescence; Morpholines; Phosphatidylinositol 3-Kinases; Phosphorylation; Polyribosomes; Protein Biosynthesis; Reverse Transcriptase Polymerase Chain Reaction; RNA; RNA, Messenger; Sepharose; Signal Transduction; Stem Cell Factor; Stem Cells; Time Factors

Phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/Akt) is thought to serve as an oncogenic signaling pathway which can be activated by Ras. The role of PI3K/Akt in Ras-mediated transformation of intestinal epithelial cells is currently not clear. Here we demonstrate that inducible expression of oncogenic Ha-Ras results in activation of PKB/Akt in rat intestinal epithelial cells (RIE-iHa-Ras), which was blocked by treatment with inhibitors of PI3K activity. The PI3K inhibitor, LY-294002, partially reversed the morphological transformation induced by Ha-Ras and resulted in a modest stimulation of apoptosis. The most pronounced phenotypic alteration following inhibition of PI3K was induction of G(1) phase cell cycle arrest. LY-294002 blocked the Ha-Ras-induced expression of cyclin D1, cyclin-dependent kinase (CDK) 2, and increased the levels of p27(kip). Both LY-294002 and wortmannin significantly reduced anchorage-independent growth of RIE-iHa-Ras cells. Forced expression of both the constitutively active forms of Raf (DeltaRaf-22W or Raf BXB) and Akt (Akt-myr) resulted in transformation of RIE cells that was not achieved by transfection with either the Raf mutant construct or Akt-myr alone. These findings delineate an important role for PI3K/Akt in Ras-mediated transformation of intestinal epithelial cells.

Topics: Androstadienes; Animals; Apoptosis; Blotting, Northern; Blotting, Western; Cell Cycle; Cell Division; Cell Line; Cell Transformation, Neoplastic; Chromones; Cyclin D1; DNA Fragmentation; Enzyme Activation; Enzyme Inhibitors; Epithelium; Flow Cytometry; Immunoblotting; Intestinal Mucosa; Intestines; Microscopy, Fluorescence; Mitogen-Activated Protein Kinases; Morpholines; Phosphoinositide-3 Kinase Inhibitors; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; ras Proteins; Rats; Sepharose; Signal Transduction; Time Factors; Transfection; Wortmannin

Occludin is an integral membrane protein of the epithelial cell tight junction (TJ). Its potential role in coordinating structural and functional events of TJ formation has been suggested recently. Using a rat salivary gland epithelial cell line (Pa-4) as a model system, we have demonstrated that occludin not only is a critical component of functional TJs but also controls the phenotypic changes associated with epithelium oncogenesis. Transfection of an oncogenic Raf-1 into Pa-4 cells resulted in a complete loss of TJ function and the acquisition of a stratified phenotype that lacked cell-cell contact growth control. The expression of occludin and claudin-1 was downregulated, and the distribution patterns of ZO-1 and E-cadherin were altered. Introduction of the human occludin gene into Raf-1-activated Pa-4 cells resulted in reacquisition of a monolayer phenotype and the formation of functionally intact TJs. In addition, the presence of exogenous occludin protein led to a recovery in claudin-1 protein level, relocation of the zonula occludens 1 protein (ZO-1) to the TJ, and redistribution of E-cadherin to the lateral membrane. Furthermore, the expression of occludin inhibited anchorage-independent growth of Raf-1-activated Pa-4 cells in soft agarose. Thus, occludin may act as a pivotal signaling molecule in oncogenic Raf- 1-induced disruption of TJs, and regulates phenotypic changes associated with epithelial cell transformation.

Topics: Animals; Cadherins; Cell Division; Cell Line, Transformed; Cell Membrane; Cell Size; Cell Transformation, Neoplastic; Claudin-1; Clone Cells; Contact Inhibition; Down-Regulation; Epithelial Cells; Humans; MAP Kinase Signaling System; Membrane Proteins; Mitogen-Activated Protein Kinases; Mutation; Occludin; Phosphoproteins; Proto-Oncogene Proteins c-raf; Rats; Retroviridae Proteins, Oncogenic; RNA, Messenger; Sepharose; Tight Junctions; Transfection; Zonula Occludens-1 Protein

Vascular TTB cells derive from murine Kaposi's sarcoma-like dermal lesions and share several phenotypic features with AIDS-associated KS spindle cells. We have recently reported that fibroblast growth factor-2 (FGF-2) promotes dramatic cytoskeletal and morphological alterations in TTB cells, concomitant with the induction of an autocrine loop for hepatocyte growth factor and a relocalization of the urokinase receptor. Since all these alterations are hallmarks of cell transformation. we attempted to verify whether FGF-2 induces a transformed phenotype in TTB cells. Our results show that FGF-2-treated TTB cells acquire the ability to grow under anchorage-independent conditions. In addition, FGF-2 markedly reduced the levels of thrombospondin-1, an antiangiogenic and tumor suppressor protein, in TTB cells. Therefore, FGF-2 induces KS-like spindle cells to acquire properties characteristic of transformed cells. This suggests that FGF-2 plays a pathogenetic role in KS not only by promoting angiogenesis, but also by conferring a transformed phenotype upon KS cells. In light of previous reports on Tat-induced release of FGF-2 into the extracellular space, our findings may provide an additional mechanism for the observed synergism between Tat and FGF-2 in the pathogenesis of KS.

Topics: Animals; Autocrine Communication; Cell Division; Cell Line, Transformed; Cell Movement; Cell Transformation, Neoplastic; Fibroblast Growth Factor 2; Gene Products, tat; HIV; Mice; Mice, Transgenic; Phenotype; Sarcoma, Kaposi; Sepharose; Skin Neoplasms; tat Gene Products, Human Immunodeficiency Virus; Thrombospondin 1; Tumor Cells, Cultured; Tumor Stem Cell Assay

The BRCA1 gene is associated with hereditary breast and ovarian cancers. BRCA1 fits the model of a classic tumor suppressor gene, a hypothesis supported by recent work demonstrating that expression of BRCA1 inhibits growth of breast and ovarian cancer cell lines. The present study was designed to test the potential of BRCA1 to reverse the transforming activity of the ras oncogene. The v-Ha ras oncogene was cloned downstream of the retrovirus LTR and stably expressed in Rat-1 cells (Rat-1/ras). Rat-1/ras (R/R) cells were fully transformed as indicated by change in morphology, colony formation in soft-agarose and tumor induction in nude mice. BRCA1 was stably expressed in R/R cells under the CMV promoter (R/R-BRCA1). The expression of ras and BRCA1 was confirmed by Western blot using monoclonal antibodies (mAbs) specific to ras and BRCA1, respectively. R/R-BRCA1 cells grew slower than the negative control, which was R/R cells transfected with vector alone (R/R-pCEP4). R/R-BRCA1 cells generated approximately 5 to 10 times less colonies in a soft-agarose assay compared to the negative control. When injected into nude mice, R/R-BRCA1 cells exhibited a delayed onset of tumorigenesis and generated smaller tumors compared to R/R or R/R-pCEP4 cells. These data strongly suggest that BRCA1 partially reverses the transforming activity of the v-Ha ras oncogene indicating that BRCA1 can bypass the effects of the v-Ha ras oncogene on cell growth. BRCA1, therefore, may be used in therapy of tumors arising due to activation of v-Ha ras oncogene.

Topics: Animals; Blotting, Western; BRCA1 Protein; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cloning, Molecular; Female; Genes, BRCA1; Genes, ras; Genetic Vectors; Humans; Mice; Mice, Nude; Neoplasm Transplantation; Promoter Regions, Genetic; ras Proteins; Rats; Sepharose; Time Factors; Up-Regulation

N-Nitroso-N-(3-keto-1,2-butanediol)-3'-nitrotyramine (NO-NTA) is a product of model browning system generated in the presence of sodium nitrite. The chemical structure of this compound has been confirmed by UV, mass and nuclear magnetic resonance, and infrared spectroscopy in our previous study. A two-stage transformation protocol was used to chemically transform the mouse embryo fibroblasts C3H10T1/2 cells. To initiate transformation, the cells were treated with benzo[a]pyrene (BaP) (0.1 mg/ml), and NO-NTA (0.01, 0.1 and 1 mg/ml) was employed subsequently to complete the transformation process. Malignant transformed foci were formed in BaP-initiated and NO-NTA promoted C3H10T1/2 cells after 8 wk. Cells treated with NO-NTA alone failed to induce transformation. However, cells initiated with BaP and promoted by cells initiated with BaP and promoted by NO-NTA demonstrated oncogenic properties. Cell lines transformed with NO-NTA-transformed colonies exhibited enhanced growth rate, anchorage independence and tumorigenicity in animals relative to parent cells. These results indicate that NO-NTA is a new tumour promoter and may induce tumour promotion by two-stage oncogenesis. Further studies on the mechanism of action of NO-NTA are now in progress.

Topics: Animals; Benzo(a)pyrene; Carcinogens; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Culture Media; Fibroblasts; Maillard Reaction; Mice; Mice, Inbred C3H; Neoplastic Stem Cells; Sepharose; Tyramine

N-Nitroso-N-(3-keto-1,2-butanediol)-3'-nitrotyramine (NO-NTA) is a product of a model browning system in the presence of sodium nitrite. In this study, the chemical structure is confirmed by spectral studies, including UV, mass spectrometry, nuclear magnetic resonance and infrared spectroscopy. NO-NTA is strongly genotoxic to the rat hepatocyte and is moderately cytotoxic to mouse C3H10T1/2 cells. Results obtained in this study indicate that NO-NTA inflicted DNA damage through the formation of a DNA adduct. In addition, C3H10T1/2 cells were treated with NO-NTA and, following addition of 12-O-tetradecanoylphorbol-13-acetate (TPA) as promotor, the increase of transformed foci indicated that NO-NTA could possibly be an initiator [corrected] of TPA tumor promotion. A transformed cell line from NO-NTA initiated and TPA promoted foci increased saturation density and growth ability in soft agar reactive to the control line. These results suggest that the formation of a genotoxic agent of nitroso-derivatives may take place in a nitrite-containing food system during processing and cooking.

Topics: Animals; Cell Division; Cell Line; Cell Survival; Cell Transformation, Neoplastic; Female; Glucose; Maillard Reaction; Mice; Mutagens; Nitrosamines; Rats; Sepharose; Sodium Nitrite; Tetrazolium Salts; Tyramine; Tyrosine

Normal human fibroblasts do not synthesize platelet-derived growth factor (PDGF), but they respond to its mitogenic action. In contrast, cells from several human fibrosarcomas have been shown to synthesize PDGF at significant levels. To investigate the possible role of PDGF expression in the development of tumors of mesenchymal origin in humans, we transfected a plasmid carrying the v-sis oncogene and a selectable marker into an infinite lifespan, non-tumorigenic human fibroblast cell strain, MSU-1.1. The v-sis gene codes for a protein homolog of PDGF-B. Of the six independent drug-resistant transfectants clonally isolated, three expressed a relatively low level of v-sis mRNA and protein, grew to a saturation density only 2-2.5 times higher than MSU-1.1 cells, did not form colonies in agar, were not growth factor independent and did not form tumors. The other three expressed v-sis mRNA and protein at a high level, grew to a very high saturation density (> 750,000 cells/cm2), replicated in medium lacking exogenous growth factors almost as rapidly as with 10% serum, formed large colonies in 0.33% agarose and, when injected into athymic mice, formed tumors that grew rapidly. Tumors that were removed after 6 weeks were classified as benign (fibromas). However, several of the tumors that were left in the animals for 6 months developed focal areas of cells showing features of poorly differentiated spindle cell or round cell sarcomas. Similarly, cells isolated from a very large sized agarose colony formed by one of the other v-sis strains expressed very high levels of v-sis mRNA and protein and formed large, very fast growing benign tumors. Re-injection of cells from the tumors yielded tumors composed of two distinct areas: spindle cell fibromas and high grade sarcomas. These results suggest that if mesenchymal cells in the body were induced to express their PDGF-B gene, this could lead to the formation of benign tumors of mesenchymal origin. The system described can serve as a model for studying the mechanisms of formation of such tumors in humans and their progression to the malignant state.

Topics: Animals; Base Sequence; Cell Adhesion; Cell Division; Cell Transformation, Neoplastic; Down-Regulation; Drug Resistance; Fibroblasts; Fibroma; Growth Substances; Humans; Karyotyping; Mice; Mice, Inbred BALB C; Mice, Nude; Molecular Sequence Data; Platelet-Derived Growth Factor; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-sis; Receptors, Platelet-Derived Growth Factor; RNA, Messenger; Sepharose; Suramin; Transfection

When mesenchyme from fetal mammary or salivary gland is implanted into adult mouse mammary gland, adjacent epithelium responds with intense hyperplasia. The hyperplastic cells are more vulnerable than are non-stimulated cells to transformation in vivo by a chemical carcinogen or by mammary tumor virus. This system provides a potentially useful model for determining how stroma contributes to mammary tumorigenesis. We have developed co-culture systems and used them to investigate in more detail the nature of the signal produced by the mesenchyme cells. Monolayers of mesenchyme cells were prepared on tissue-culture wells. The mesenchyme cells were trapped on the surface by a thin overlay of agarose. Primary mammary epithelial cells were cultured atop this barrier layer, either as organoids in collagen gels for assessment of anchorage-dependent growth, or as single-cell dispersions in soft agarose for assessment of anchorage-independent growth. Our procedures for assay of anchorage-independent growth allow us for the first time to detect and measure this transformation-defining characteristic in non-immortalized mammary epithelial cells in primary culture. Fetal mammary fat pad precursor tissue and fetal salivary mesenchyme both stimulated anchorage-dependent growth of mammary epithelium, with cell number increasing as much as fifteenfold during a 6-day culture period. These same fetal tissues also stimulated anchorage-independent growth of the mammary epithelial cells, with colony-forming efficiencies of up to 40% in co-cultures with salivary mesenchyme. No colonies formed in the absence of mesenchyme. Cells of colonies contained keratin, which indicates that the colonies grew from epithelial cells and not from a contaminant of another cell type. When co-cultured epithelial cells were subsequently re-cultured in the absence of mesenchyme, they lost their ability to grow independent of anchorage. No colonies grew in co-cultures with fetal cells from heart, kidney, or lung, which is consistent with the lack of stimulation by these tissues in the mammary gland in vivo. A tumor promoter, 12-O-tetradecanoylphorbol acetate (TPA), also caused anchorage-independent growth of the dispersed mammary epithelial cells. Culture medium conditioned by primary or early-passage salivary mesenchyme cells was capable of stimulating growth under both anchorage-dependent and anchorage-independent conditions, confirming that these effects are mediated by a paracrine factor. The re

Topics: Animals; Cell Transformation, Neoplastic; Culture Techniques; Cytological Techniques; Epithelial Cells; Epithelium; Fetal Tissue Transplantation; Mammary Glands, Animal; Mesoderm; Mice; Mice, Inbred BALB C; Phenotype; Sepharose; Stem Cells; Stromal Cells; Tetradecanoylphorbol Acetate

Using affinity chromatography on ATP-agarose, we have identified a major ATP-binding protein in Nonidet P-40 extracts of avian and mammalian cells labeled with [35S]methionine. After washing ATP-agarose beads with high-ionic-strength buffer (0.4 M NaCl), the 37-kD protein was shown to be one of the major ATP-binding proteins while p72 and grp78, which are members of the hsp70 family, also bound to ATP-agarose. This protein consisted of several spots on two-dimensional gel electrophoresis. The isoelectric point of the most basic spot was approximately 9.2 in chick embryo fibroblasts, whereas it was about 8.8 in mouse 3T3 cells. The identities of these proteins in mouse and chick cells were confirmed by peptide mapping. After heat-shock treatment of BALB/3T3 cells, the major heat-shock protein, hsp70, was shown to be induced very rapidly after heat shock and was recovered in the ATP-binding fraction. Besides hsp70, a 37-kD protein was also found to be induced by heat shock. This protein was drastically induced by treating the cells with alpha,alpha'-dipyridyl, an iron chelating reagent, but not with sodium arsenite, calcium ionophore, or tunicamycin. The synthesis and the total amount of this ATP-binding protein increased in mouse 3T3 cells transformed by simian virus 40, methylcholanthrene, or activated c-Ha-ras oncogene compared to their normal counterparts. The incorporation of [32P]orthophosphate was not detected in either normal or transformed cells. These studies established that a major ATP-binding protein of Mr = 37,000 is a heat-inducible protein and that the synthesis of this protein is regulated by malignant transformation.

Topics: 2,2'-Dipyridyl; Adenosine Triphosphate; Animals; Arsenic; Arsenites; Calcimycin; Carrier Proteins; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cells, Cultured; Chick Embryo; Electrophoresis, Polyacrylamide Gel; Endoplasmic Reticulum Chaperone BiP; Fibroblasts; Heat-Shock Proteins; Methionine; Mice; Mice, Inbred BALB C; Precipitin Tests; Sepharose; Sulfur Radioisotopes; Tetradecanoylphorbol Acetate; Tunicamycin

This paper describes the evaluation of a colony formation assay using automated image analysis, which permits the tracking of growth at the individual colony level, such that a growth rate can be estimated for each colony followed. In principle, this will permit quantitative characterization of cellular heterogeneity in growth rate and cellular heterogeneity in response to proliferation-modifying agents. In addition, we have demonstrated the possibility of using correlative microscopy to relate growth rate to other parameters, using metabolic viability as an example. This should be useful for determining cellular characteristics associated with proliferative behavior and response to proliferation-modifying agents.

Topics: Adenocarcinoma; Cell Transformation, Neoplastic; Humans; Ileal Neoplasms; Image Processing, Computer-Assisted; Sepharose; Tumor Cells, Cultured

Differences in tumorigenicity between the Epstein-Barr virus (EBV) genome-negative and -positive epithelial A2L/AH hybrid cell lines have been described recently. Further differences in growth characteristics of the two epithelial hybrid cells have since been examined. Differences were found in doubling time and cloning efficiency in agarose of the two cell lines. In addition, the EBV genome-negative epithelial hybrid cell line (cl-654), unlike the EBV genome-positive epithelial hybrid cell line (cl-1), failed to grow in the unmanipulated nude mice, but was successfully transplanted in 60Co-irradiated nude mice. The histopathologic findings of tumors induced by the inoculation of cl-654 cells in nude mice showed more differentiated carcinoma than the findings of tumors induced by cl-1 cells. The data suggest that EBV is associated with an alteration of cell differentiation in epithelial cells as well as tumorigenicity in nude mice.

Topics: Animals; Carcinoma; Cell Division; Cell Line; Cell Transformation, Neoplastic; Clone Cells; Culture Media; Epithelial Cells; Female; Genes, Viral; Herpesvirus 4, Human; Hybrid Cells; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; Sepharose

Our aim was to determine if the selection of human tumor cells with enhanced anchorage-independent growth capacity was associated with alterations in methionine auxotrophy. Cells with an increased ability to form colonies on soft agarose were selected from human melanoma (MeWo) and neuroepithelioma (SK-N-MC) cell lines. In contrast to their respective parental lines, a high proportion of the agarose-selected variants were completely unable to proliferate in methionine-free medium containing its immediate precursor homocysteine. The variants exhibited no significant change in their total DNA 5-methylcytosine content and showed no stimulation of either RNA or DNA synthesis upon the addition of homocysteine when the cells were cultured in methionine-free medium. These variants were unable to synthesize [3H]S-adenosylmethionine from [3H]adenine and homocysteine. The failure to detect the accumulation of [3H]S-adenosylmethionine in these variant lines was not likely due to the enhanced turnover of S-adenosylmethionine but rather to a reduced ability to synthesize methionine from homocysteine and 5-methyltetrahydrofolic acid. These results support our hypothesis that alterations in the metabolism of methionine and/or intracellular transmethylating activities may contribute to, or be associated with, the autonomous growth of malignant human tumor cells.

Topics: Cell Line; Cell Transformation, Neoplastic; Genetic Variation; Homocysteine; Humans; Melanoma; Methionine; Neuroblastoma; Nucleic Acids; S-Adenosylmethionine; Sepharose; Tumor Cells, Cultured

In these experiments individual colonies growing in agarose seeded with monocellular suspensions from freshly disaggregated naturally-occurring mouse mammary tumours, induced by the murine mammary tumour virus (MMTV), were reimplanted into mammary fat pads of virus-free mice. It was found that only a small proportion of these colonies generated tumours and that the implantation of multiple colonies in each site did not result in disproportionate, synergistic, increase in tumour takes. It was also observed that the proportion of colonies which were tumourigenic on reimplantation differed for each donor tumour and represented only a small fraction of the total cell population (0.001%-0.1%). However, this value was significantly higher in tumours which produced large numbers of deposits in lung colony assays following i.v. injections, than in tumours of low pulmonary colonisation potential. A point of particular interest was that tumours derived from agarose colonies of spontaneously metastatic donor tumours were substantially more spontaneously metastatic themselves than those from nonmetastatic donors, indicating that this property is heritable through numerous cell divisions, manipulations in vitro and transplantation procedures. From these results it is concluded that measurement of clonogenicity in agar is useful as an index of the capability of a tumour to propagate itself and to colonise new sites, but that individual agarose colonies are not all the progeny of potentially immortal stem cells.

Topics: Animals; Cell Transformation, Neoplastic; Mammary Neoplasms, Experimental; Mice; Mice, Inbred C3H; Sepharose; Tumor Cells, Cultured; Tumor Stem Cell Assay

To investigate the basic mechanisms of granuloma formation, in vitro granulomas were induced by culturing murine spleen cells in the presence of artificial microparticles. Large granulomas developed around dextran beads. The lesions were inducible by spleen cells from either normal mice or athymic nude mice. Minimal inflammation was produced around latex beads. The histologic features and time kinetics of granulomas in vitro. Culture supernatants of dextran induced granulomas contained high levels of interleukin-1 (IL-1) activity but not interleukin-2 (IL-2) or interleukin-4 (IL-4) activity. IL-1 activity was correlated with granuloma size. Additionally, granulomas were produced by culturing spleen cells in the presence of agarose beads coupled to recombinant IL-1 or recombinant tumor necrosis factor-alpha (TNF-alpha). Granulomatous lesions also were induced by macrophage-enriched populations in the presence of monokine-coupled beads. Adherent macrophages, but not nonadherent cells, were required for induction of granulomas in vitro. In contrast, very small lesions were seen when spleen cells or adherent cells were cultured in the presence of beads coupled to recombinant IL-2 or recombinant interferon-gamma (IFN-gamma). These results suggest that macrophages and monokines such as IL-1 and TNF-alpha play an essential role in granuloma formation in vitro.

Topics: Animals; Biological Factors; Cell Transformation, Neoplastic; Cells, Cultured; Cytokines; Dextrans; Female; Granuloma; Immunosuppressive Agents; Interleukin-1; Macrophages; Mice; Mice, Inbred Strains; Microspheres; Monokines; Polystyrenes; Sepharose; Spleen; Tumor Necrosis Factor-alpha

Platelet derived growth factor cooperated with middle T antigen in inducing growth in agarose medium of secondary cultured rat embryo cells transfected with a polyoma virus middle T antigen cDNA clone. In contrast, epidermal growth factor and a conditioned medium containing transforming growth factor did not stimulate the colony-forming efficiency of such cells in the agarose medium.

Topics: Animals; Antigens, Polyomavirus Transforming; Antigens, Viral, Tumor; Cell Transformation, Neoplastic; Cells, Cultured; Epidermal Growth Factor; Female; Fibroblasts; Oncogene Proteins, Viral; Peptides; Platelet-Derived Growth Factor; Polyomavirus; Pregnancy; Rats; Rats, Inbred F344; Sepharose; Transfection; Transforming Growth Factors

Primate neoplastic and finite cell lines were tested in one in vivo and two in vitro test systems: adult nude mice, muscle organ culture (MOC) and soft agarose (SA). Comparison of the sensitivity of the systems indicated that nude mice were inferior to either in vitro system: WI-38 VA13 (an SV40 transformed cell line) did not cause tumours in these animals yet it behaved as if it were neoplastic in MOC and formed colonies in SA. There was complete correlation between results obtained in MOC and SA. All cell lines which produced tumors in vivo were positive in both in vitro test systems. None of the lines which showed normal patterns in MOC and in SA was tumorigenic in nude mice. Since testing in vitro is simpler, faster, and is thought to be reliable, we recommend SA followed by MOC as the initial assays for determining tumorigenicity of cells.

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Female; Humans; Mice; Mice, Nude; Muscles; Neoplasm Transplantation; Neoplasms; Organ Culture Techniques; Sepharose

In efforts to determine common and consistent morphological parameters of transformation in C3H/10T1/2 cells, 15 cell lines transformed by different carcinogens were examined with the scanning electron microscope (SEM) and compared to non-transformed cells. Cell lines were studied at different passages, at different cell densities, and after growth as anchorage dependent or independent cultures. The transformed cell lines could be distinguished in the SEM from non-transformed cultures by the expression of one or more of the following morphological characteristics: formation of mini- or macro-foci (random piling of cells on top of each other), pleiomorphism in cell size and shape, and cell surface complexity. The extent to which these characteristics were expressed varied widely in the different transformed cell lines. Light microscopic scoring for different types of foci also revealed broad variability among the different transformed lines. At the SEM level, cell lines could not be characterized as transformed on an individual cell basis. However, all transformed cell lines could be definitively characterized as transformed on a population basis due to the presence of mini-foci. The various transformed cell lines were classified semi-quantitatively into categories based on the extent of expression of the different morphological characteristics. There was broad correspondence between the morphological classification and the relative plating efficiencies of the cell lines in soft agarose.

Topics: Animals; Cell Transformation, Neoplastic; Cells, Cultured; Embryo, Mammalian; Mice; Mice, Inbred C3H; Microscopy, Electron, Scanning; Sepharose

At the light of the increasing evidence that malignant transformation is a multistep process the wide correlation between growth in soft agar and various degrees of tumorigenicity suggests that this could be a property of the early stages of malignant transformation. In the present paper we give evidence indicating that colony formation in agarose gels containing deoxyglucose can operate as a more restrictive criterium than growth in soft agar for malignant transformation in vitro, possibly associated to advanced stages in this process.

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Cricetinae; Culture Media; Deoxyglucose; Kinetics; Mesocricetus; Sepharose

Differentially L-fucose-labelled glycopeptides from the surface of a Syrian golden hamster (SGH) fetal lung control cell line were compared with those from chemically-transformed and tumour cell lines derived from the control line by cochromatography on Concanavalin A-Sepharose (Con A-Sepharose) and Sephadex G-50. Quantitative differences were found both in the unbound and specifically-bound fractions between control and transformed cells upon Con A-Sepharose chromatography. In the glycopeptides from transformed and tumour cells, the unretarded fraction was concomitantly decreased compared to the controls. When the ratio of unbound to specifically-bound fractions was used, a statistically significant difference could be calculated between the values of control versus transformed or tumour cells. In all transformed and tumour cell lines investigated, the quantitative change in Concanavalin A binding, expressed as an increase of the ratio of unretarded to specifically-bound glycopeptides, was paralleled by a shift of transformed or tumour glycopeptides to higher apparent molecular weight compared to the control in gel chromatography.

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Concanavalin A; Cricetinae; Fetus; Glycopeptides; Lung; Membrane Proteins; Mesocricetus; Neoplasms, Experimental; Sepharose

A method for the in vitro identification of transformed rat liver epithelial and hepatoma cells was developed using the preferential microagglutination of concanavalin A (Con A) coupled to agarose beads (Con A:agarose) to their colonies. Con A:agarose attaches to the cell surface through a specific interaction of the Con A moiety and its receptors. The attachment is dependent on the mobility and aggregation of the Con A: receptor complex on the membrane. Agents which interfere with the interaction reduced the bead density over the colonies. For the quantitative determination of transformed colonies in a mixed-cell population, also containing untransformed cells, it is essential to compare colonies of a similar size or to use the bead density per unit area as the index. When a variety of rat liver epithelial cell lines were tested, the assay proved to be simple, reproducible, and precise. It was found that the increased attachment of Con A:agarose to cell colonies is a characteristic of transformed or malignant rat liver cells.

Topics: Animals; Cell Transformation, Neoplastic; Cells, Cultured; Concanavalin A; Liver; Liver Neoplasms, Experimental; Liver Regeneration; Rats; Sepharose

At the present time, growth in agar suspension is one of the best in vitro correlates of tumorigenicity. Growth in agarose, however, has not been evaluated extensively as an in vitro criterion for tumorigenicity. In the present study we have tested 19 cell lines, including six mouse-human hybrids, for growth in agarose and agar in the presence and absence of exogenous hypoxanthine. None of the six nontumorigenic cell lines grew in agar or agarose. Ten of the 13 tumorigenic cell lines grew in both agar and agarose with about equal efficiency. The remaining three tumorigenic cell lines grew well in agarose but poorly or not at all in agar. Hypoxanthine did not stimulate the growth in agar or agarose of any of the cell lines except BHK. We conclude that growth in agarose may be a more sensitive marker for tumorigenicity than growth in agar and that BHK is exceptional in its sensitivity to supplemental purines.

Topics: Agar; Animals; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cricetinae; HeLa Cells; Humans; Hybrid Cells; Hypoxanthines; Kidney; Mice; Neoplasm Transplantation; Polysaccharides; Sepharose

Normal C57 Black mouse embryo cells did not form colonies in agarose, but rare variant (ar+) cells able to grow in agarose were detected. Fluctuation analysis showed that ar+ variants arose by spontaneous mutation in the cultured cells. The frequency of ar+ variants was increased by treating cells with N-methyl-N'nitro-N-nitrosoguanidine or ethyl methane sulphonate, or by abortive infection by human adenovirus type 5. Induced ar+ cells were fibroblastic; most grew slowly and had slightly reduced saturation density and increased serum requirement, but formed colonies in agarose. Fourteen of twenty ar+ clones induced by Ad5 were T antigen negative and two of these were also negative when tested for viral DNA. Six clones contained a few cells that were T antigen positive when first tested, but were negative when retested later. The ar+ variants were tumorigenic in athymic and in normal syngeneic mice. The results suggest that the ar+ phenotype can arise by spontaneous or chemically-induced mutation, and can be induced by adenovirus by a process different from classical transformation.

Topics: Adenoviruses, Human; Animals; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cells, Cultured; Culture Media; Ethyl Methanesulfonate; Fibroblasts; Genetic Variation; Methylnitronitrosoguanidine; Mice; Mutagens; Mutation; Phenotype; Polysaccharides; Sepharose

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Cells, Cultured; Chromosomes; Cricetinae; Culture Media; Embryo, Mammalian; Fibroblasts; Glucosephosphate Dehydrogenase; Isoenzymes; Mesocricetus; Methoxsalen; Mice; Mice, Inbred Strains; Neoplasms, Experimental; Phenotype; Rats; Sepharose; Ultraviolet Rays

The influence of population density in the progression from the nonneoplastic to the neoplastic state has been reassessed. Two twice-cloned, nonneoplastic mouse lines, NCTC 7914 and 7915, were transferred each 3 to 4 days at inoculum sizes selected to minimize or maximize cell-cell contact, 1 X 10(5) or 4 X 10(5) cells/T-15, respectively. As tested by in vivo assay, the regime designed to minimize cell-cell contact did not reproducibly delay transformation, and tumor production was observed in all lines, irrespective of inoculum size. Also, results of tumorigenesis assays correlated with blind evaluation of morphological and cytological alterations, growth in agarose, and susceptibility to killing by activated macrophages. Generally higher saturation densities were seen as a function of period in culture, and no significant differences in glucose utilization or lactic acid production were observed between nonneoplastic and neoplastic cell populations.

Topics: BCG Vaccine; Cell Adhesion; Cell Communication; Cell Count; Cell Line; Cell Transformation, Neoplastic; Contact Inhibition; Cytotoxicity, Immunologic; Glycolysis; Macrophages; Mycobacterium bovis; Sepharose

Topics: Animals; Cell Transformation, Neoplastic; Culture Media; Mice; Mutation; Neoplasm Transplantation; Neoplasms, Experimental; Sepharose; Time Factors

Human hematopoietic cell lines, which had been classified on the basis of studies on clonality, and morphological, chromosomal and functional parameters as lymphoblastoid cell lines (LCL) of presumed non-neoplastic origin, and lymphoma, myeloma and leukemia lines of proven malignant origin, were tested for tumorigenic potential on subcutaneous transplantation to nude mice and for capacity to grow in semi-solid medium in vitro. Recently established LCL failed to grow both in nude mice and in agarose. In contrast, some of the LCL which had developed secondary chromosomal alterations during continuous cultivation for periods exceeding several years were tumorigenic and/or had the capacity to form colonies in agarose. Most lymphoma lines formed colonies in agarose and tumors in the mice. One of the two myeloma lines formed subcutaneous tumor which, however, showed no progressive growth. The other myeloma line failed to grow. Both myeloma lines, however, formed colonies in agarose. The myeloid leukemia line was tumorigenic while two of the three tested lymphocytic leukemia lines failed to grow in the mice. All leukemia lines formed colonies in agarose. We conclude from this study that: (1) Of the two types of Epstein-Barr virus containing cell lines [LCL and Burkitt lymphoma (BL) lines], only BL lines were shown to form tumors when inoculated subcutaneously in nude mice and had the capacity to grow in agarose in vitro. This shows that EBV transformation per se does not necessarily render lymphocytes tumorigenic in nude mice. The capacity to form colonies in agarose is not acquired either. (2) Changes of the karyotype and several phenotypic characteristics which occur in the originally diploid LCL during prolonged cultivation in vitro may be accompanied by the acquisition of the potential to grow subcutaneously in nude mice and in agarose in vitro. (3) The inconsistency with regard to the capacity of come of the neoplastic cell lines to grow in nude mice or in agarose seems to underline that neither of the two tests is a reliable criterion for malignancy of human lymphoma, leukemia and myeloma cell lines.

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Cells, Cultured; Clone Cells; Hematopoietic Stem Cell Transplantation; Hematopoietic Stem Cells; Herpesvirus 4, Human; Humans; Lymphocyte Transfusion; Lymphocytes; Mice; Mice, Nude; Neoplasms, Experimental; Sepharose; Transplantation, Heterologous

Adherent fibroblast-like cells from paired lines, one non-neoplastic and the other "spontaneously" transformed neoplastic, were compared in simultaneous in vivo and in vitro assays. The in vivo assay was the i.m. implantation of 10(6) or 10(7) cells in irradiated syngeneic animals, and the two in vitro assays were the evaluation of colony morphology on plastic and the enumeration of colony growth in semisolid agarose. The percentage of colonies diagnosed from their morphology as neoplastic correlated with tumorigenicity as follows: 100% always indicated a tumorigenic cell population with tumor latent periods from 6 to 230 days and tumor incidence from 40 to 100%; 0% always indicated a nontumorigenic cell population; 1 to 32% indicated either a tumorigenic cell line with long tumor latent period (218 days) with 70% tumor incidence or a nontumorigenic cell line. Growth in agarose, as measured by colony number and size, correlated with tumorigenicity as follows: nontumorigenic cell lines produced no colonies; tumorigenic cell lines produced colonies, but not always larger than 0.1 mm in diameter. The number of size or colonies did not correlate with the tumor latent period or tumor incidence. Therefore, both in vitro tests were reliable qualitative assays of spontaneous neoplastic transformation, but they did not correlate directly with the tumor incidence or mean tumor latent period. The relative success of the agarose assay emphasizes the importance of decreased anchorage dependence for progressive growth of injected cells as a malignant neoplasm in vivo.

Topics: Animals; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cells, Cultured; Cricetinae; Mice; Plastics; Rats; Sepharose; Time Factors

Following treatment of Syrian hamster embryo cells with benzo(a)pyrene, the time required for the expression of enhanced fibrinolytic activity was examined. For this study, the fibrin-agarose overlay method was developed to distinguish the activity of normal and transformed colonies of hamster cells. Colonies possessing enhanced fibrinolytic activity were not observed one passage (2 weeks after treatment). Morphologically transformed colonies, which exhibited no enhanced fibrinolytic activity, were observed 8 days following treatment. In contrast to these two early changes, cells capable of growth in soft agar were observed much later (6 to 8 weeks after treatment). Untreated Syrian hamster embryo cells generally senesced and did not exhibit enhanced fibrinolytic activity. Approximately 1 of 10 untreated cultures escaped senescence and evolved as a continuous cell line; such cultures frequently exhibited enhanced fibrinolytic activity. These results suggest that the acquisition of enhanced fibrinolytic activity, while perhaps not a cause of neoplastic transformation, may reflect a loss of control of the normal function of the cellular genetic apparatus during the process of transformation.

Topics: Benzopyrenes; Cell Transformation, Neoplastic; Cells, Cultured; Clone Cells; Culture Media; Fibrin; Fibrinolysis; Sepharose; Time Factors

Topics: Animals; Cell Division; Cell Line; Cell Membrane; Cell Transformation, Neoplastic; Culture Media; DNA; Fibroblasts; Glycoproteins; Mice; Mice, Inbred BALB C; Mutation; Peptide Hydrolases; Protein Biosynthesis; RNA, Ribosomal; Sepharose; Simian virus 40; Temperature

Topics: Alpharetrovirus; Animals; Antimetabolites; Avian Leukosis Virus; Avian Sarcoma Viruses; Caseins; Cell Transformation, Neoplastic; Chick Embryo; Chromatography, Affinity; Culture Media; Enzyme Activation; Fibroblasts; Peptide Hydrolases; Plasminogen; Protease Inhibitors; Sepharose; Species Specificity