agar and Cell-Transformation--Neoplastic

The technique of bone marrow cultures has been shown to be of value in childhood acute leukemia. It now appears that acute myelogenous leukemia may be due to defective maturation of normal progenitor cells. The pattern of growth of these cells has been demonstrated to be of prognostic value. In contrast, the growth of normal progenitor cells from the bone marrow cultures of children with acute lymphocytic leukemia (ALL) may be due to the few remaining normal cells. The cause of granulocytopenia in childhood ALL is still unclear.

Topics: Acute Disease; Agar; Bone Marrow; Cell Transformation, Neoplastic; Cells, Cultured; Child; Child, Preschool; Colony-Stimulating Factors; Culture Media; Granulocytes; Hematopoiesis; Humans; Leukemia; Leukemia, Lymphoid; Leukemia, Myeloid, Acute; Phytohemagglutinins

Topics: Agar; Cell Transformation, Neoplastic; Cells, Cultured; Chromosomes, Human, 21-22 and Y; Hematopoietic Stem Cells; Humans; Leukemia, Myeloid; Time Factors

The progression of anchorage-dependent epithelial cells to anchorage-independent growth represents a critical hallmark of malignant transformation. Using an in vitro model of human papillomavirus (HPV)-induced transformation, we previously showed that acquisition of anchorage-independent growth is associated with marked (epi)genetic changes, including altered expression of microRNAs. However, the laborious nature of the conventional growth method in soft agar to measure this phenotype hampers a high-throughput analysis. We developed alternative functional screening methods using 96- and 384-well ultra-low attachment plates to systematically investigate microRNAs regulating anchorage-independent growth. SiHa cervical cancer cells were transfected with a microRNA mimic library (n = 2019) and evaluated for cell viability. We identified 84 microRNAs that consistently suppressed growth in three independent experiments. Further validation in three cell lines and comparison of growth in adherent and ultra-low attachment plates yielded 40 microRNAs that specifically reduced anchorage-independent growth. In conclusion, ultra-low attachment plates are a promising alternative for soft-agar assays to study anchorage-independent growth and are suitable for high-throughput functional screening. Anchorage independence suppressing microRNAs identified through our screen were successfully validated in three cell lines. These microRNAs may provide specific biomarkers for detecting and treating HPV-induced precancerous lesions progressing to invasive cancer, the most critical stage during cervical cancer development.

Topics: Agar; Alphapapillomavirus; Cell Transformation, Neoplastic; Female; Humans; MicroRNAs; Papillomaviridae; Papillomavirus Infections; Uterine Cervical Neoplasms

For the last five decades, measuring the ability of cells to grow in soft agar has served as the gold standard assay for in vitro cellular transformation. Nevertheless, the soft agar colony formation assay is time consuming and ill-suited for high-throughput screens. This unit describes an equally qualitative and quantitative assay known as growth in low attachment or GILA. The GILA assay is suitable for high-throughput pharmacological or genetic screens and allows the simultaneous examination of multiple cell lines and experimental perturbations. GILA conditions are specific and relevant to the transformed state because they depend on a property of cancer cells that is not shared by non-transformed cells. The GILA assay enables ex vivo drug sensitivity testing of patient-derived tumor cells to define precise treatments for individual patients. © 2016 by John Wiley & Sons, Inc.

Topics: Agar; Cell Adhesion; Cell Culture Techniques; Cell Separation; Cell Transformation, Neoplastic; Drug Screening Assays, Antitumor; Genetic Testing; High-Throughput Screening Assays; Humans; Neoplasms; Tumor Cells, Cultured

Colony formation in soft agar is the gold-standard assay for cellular transformation in vitro, but it is unsuited for high-throughput screening. Here, we describe an assay for cellular transformation that involves growth in low attachment (GILA) conditions and is strongly correlated with the soft-agar assay. Using GILA, we describe high-throughput screens for drugs and genes that selectively inhibit or increase transformation, but not proliferation. Such molecules are unlikely to be found through conventional drug screening, and they include kinase inhibitors and drugs for noncancer diseases. In addition to known oncogenes, the genetic screen identifies genes that contribute to cellular transformation. Lastly, we demonstrate the ability of Food and Drug Administration-approved noncancer drugs to selectively kill ovarian cancer cells derived from patients with chemotherapy-resistant disease, suggesting this approach may provide useful information for personalized cancer treatment.

Topics: Adenosine Triphosphate; Agar; Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cell Transformation, Neoplastic; Chemistry, Pharmaceutical; Drug Design; Drug Screening Assays, Antitumor; Female; Fibroblasts; Flow Cytometry; High-Throughput Screening Assays; Humans; Open Reading Frames; Ovarian Neoplasms

Previous work has shown that the Simian Virus 40 T antigen (T antigen) cannot transform mouse embryo fibroblasts (MEFs) that do not express the type 1 insulin-like growth factor receptor (IGF-IR). We have now investigated the mechanism(s) by which the transforming activity of T antigen is affected by IGF-IR signaling. We demonstrate that transformation by T antigen of MEFs and several other cell lines requires an insulin receptor substrate-1 (IRS-1) phosphorylated on tyrosines. If IRS-1 is not expressed, or is serine phosphorylated or otherwise inactive, T antigen fails to transform cells in culture. For instance, while T antigen cannot transform 32D myeloid cells (that do not express IRS-1), its transforming activity is restored by the expression of a wild-type IRS-1, but not of an IRS-1 mutated at the PI3K binding sites. The importance of IRS-1 activation of PI3K in T-antigen transformation is supported by the finding that a constitutively activated p110 subunit of PI3K, a target of IRS-1, overcomes the inability of T antigen to transform MEFs with a serine phosphorylated IRS-1. Taken together, these results indicate that the IRS-1/PI3K signaling is one of the mechanisms regulating transformation by the SV40 T antigen. We propose that the requirement for a tyrosyl-phosphorylated IRS-1 provides a mechanism to explain the failure of T antigen to transform MEFs with deleted IGF-IR genes.

Topics: Agar; Animals; Antigens, Polyomavirus Transforming; Antigens, Viral, Tumor; Binding Sites; Blotting, Western; Breast Neoplasms; Cell Line; Cell Line, Transformed; Cell Survival; Cell Transformation, Neoplastic; Cells, Cultured; Fibroblasts; Gene Deletion; Insulin Receptor Substrate Proteins; Mice; Mutation; Neurons; Phosphatidylinositol 3-Kinases; Phosphoproteins; Phosphorylation; Pol1 Transcription Initiation Complex Proteins; Receptor, IGF Type 1; Ribosomes; RNA; RNA, Ribosomal; Serine; Signal Transduction; Time Factors; Transfection; Tyrosine

The c-Myc oncoprotein plays a central role in human cancer via its ability to either activate or repress the transcription of essential downstream targets. For many of the repressed target genes, down-regulation by c-Myc relies on its ability to bind and inactivate the transcription factor Miz-1. Although Miz-1 inactivation is suspected to be essential for at least some of the biological activities of c-Myc, it has been difficult to demonstrate this requirement experimentally. Using a combination of short hairpin RNA-mediated knockdown and a previously characterized mutant of c-Myc that is defective for Miz-1 inactivation, we examined whether this inactivation is critical for three of the most central biological functions of c-Myc, cell cycle progression, transformation, and apoptosis. The results of this analysis demonstrated that in the in vitro assays utilized here, Miz-1 inactivation is dispensable for c-Myc-induced cell cycle progression and transformation. In marked contrast, the ability of c-Myc to induce apoptosis in primary diploid human fibroblasts in response to growth factor withdrawal is entirely dependent on its ability to inactivate Miz-1. These data have a significant impact on our understanding of the biochemical mechanisms dictating how c-Myc mediates opposing biological functions, such as transformation and apoptosis, and demonstrate the first requirement for Miz-1 inactivation in any of the biological functions of c-Myc.

Topics: Agar; Animals; Apoptosis; Blotting, Western; Cell Cycle; Cell Line; Cell Transformation, Neoplastic; DNA-Binding Proteins; Down-Regulation; Humans; Immunoblotting; Immunoprecipitation; Kruppel-Like Transcription Factors; Mutation; Plasmids; Polymerase Chain Reaction; Protein Binding; Proto-Oncogene Proteins c-myc; Rats; Retroviridae; Reverse Transcriptase Polymerase Chain Reaction; RNA; Transcription Factors; Transcription, Genetic; Transfection

Cdc42, a Ras-related GTP-binding protein, has been implicated in the regulation of the actin cytoskeleton, membrane trafficking, cell-cycle progression, and malignant transformation. We have shown previously that a Cdc42 mutant (Cdc42(F28L)), capable of spontaneously exchanging GDP for GTP (referred to as "fast-cycling"), transformed NIH 3T3 cells because of its ability to interfere with epidermal growth factor receptor (EGFR)-Cbl interactions and EGFR down-regulation. To further examine the link between the hyperactivation of Cdc42 and its ability to alter EGFR signaling and thereby cause cellular transformation, we examined the effects of expressing different forms of the Cdc42-specific guanine nucleotide exchange factor, intersectin-L, in fibroblasts. Full-length intersectin-L exhibited little ability to stimulate nucleotide exchange on Cdc42, whereas a truncated version that contained five Src homology 3 (SH3) domains, the Dbl and pleckstrin homology domains (DH and PH domains, respectively), and a C2 domain (designated as SH3A-C2) showed modest guanine nucleotide exchange factor activity, whereas a form containing just the DH, PH, and C2 domains (DH-C2) strongly activated Cdc42. However, DH-C2 showed little ability to stimulate growth in low serum or colony formation in soft agar, whereas SH3A-C2 gave rise to a much stronger stimulation of cell growth in low serum and was highly effective in stimulating colony formation. Moreover, although SH3A-C2 strongly transformed fibroblasts, it differed from the actions of the Cdc42(F28L) mutant, as SH3A-C2 showed little ability to alter EGFR levels or the lifetime of EGF-coupled signaling through ERK. Rather, we found that SH3A-C2 exhibited strong transforming activity through its ability to mediate cooperation between Ras and Cdc42.

Topics: Actins; Adaptor Proteins, Vesicular Transport; Agar; Animals; cdc42 GTP-Binding Protein; Cell Cycle; Cell Line; Cell Line, Transformed; Cell Transformation, Neoplastic; COS Cells; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Fibroblasts; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Immunoblotting; Immunoprecipitation; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase 4; Mice; Microscopy, Fluorescence; Mitogen-Activated Protein Kinase Kinases; Models, Biological; Mutation; NIH 3T3 Cells; Phosphatidylinositol 3-Kinases; Plasmids; Protein Binding; Protein Structure, Tertiary; ras Proteins; Signal Transduction; src Homology Domains; Time Factors; Transfection

The apoptosis-promoting protein Par-4 has been shown to be down-regulated in Ras-transformed NIH 3T3 fibroblasts through the Raf/MEK/ERK MAPK pathway. Because mutations of the ras gene are most often found in tumors of epithelial origin, we explored the signaling pathways utilized by oncogenic Ras to down-regulate Par-4 in RIE-1 and rat ovarian surface epithelial (ROSE) cells. We determined that constitutive activation of the Raf, phosphatidylinositol 3-kinase, or Ral guanine nucleotide exchange factor effector pathway alone was not sufficient to down-regulate Par-4 in RIE-1 or ROSE cells. However, treatment of Ras-transformed RIE-1 or ROSE cells with the MEK inhibitors U0126 and PD98059 increased Par-4 protein expression. Thus, although oncogenic Ras utilizes the Raf/MEK/ERK pathway to down-regulate Par-4 in both fibroblasts and epithelial cells, Ras activation of an additional signaling pathway(s) is required to achieve the same outcome in epithelial cells. Methylation-specific PCR showed that the par-4 promoter is methylated in Ras-transformed cells through a MEK-dependent pathway and that treatment with the DNA methyltransferase inhibitor azadeoxycytidine restored Par-4 mRNA transcript and protein levels, suggesting that the mechanism for Ras-mediated down-regulation of Par-4 is by promoter methylation. Support for this possibility is provided by our observation that Ras transformation was associated with up-regulation of Dnmt1 and Dnmt3 DNA methyltransferase expression. Finally, ectopic Par-4 expression significantly reduced Ras-mediated growth in soft agar, but not morphological transformation, highlighting the importance of Par-4 down-regulation in specific aspects of Ras-mediated transformation of epithelial cells.

Topics: Agar; Alleles; Animals; Apoptosis; Apoptosis Regulatory Proteins; Azacitidine; Blotting, Northern; Blotting, Western; Butadienes; Cell Line; Cell Line, Tumor; Cell Transformation, Neoplastic; Decitabine; DNA (Cytosine-5-)-Methyltransferase 1; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; DNA Methyltransferase 3A; DNA, Complementary; Down-Regulation; Enzyme Inhibitors; Epithelial Cells; Female; Fibroblasts; Flavonoids; Genetic Vectors; Humans; Intracellular Signaling Peptides and Proteins; Mice; Mutation; NIH 3T3 Cells; Nitriles; Ovary; Phosphatidylinositol 3-Kinases; Polymerase Chain Reaction; raf Kinases; Rats; RNA; RNA, Messenger; Signal Transduction; Up-Regulation

Chlorogenic acid, the ester of caffeic acid with quinic acid, is one of the most abundant polyphenols in the human diet. The antioxidant and anticarcinogenic properties of chlorogenic acid have been established in animal studies. However, little is known about the molecular mechanisms through which chlorogenic acid inhibits carcinogenesis. In this study, we found that chlorogenic acid inhibited the proliferation of A549 human cancer cells in vitro. The results of the soft agar assay indicated that chlorogenic acid suppressed 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced neoplastic transformation of JB6 P+ cells in a dose-dependent manner. Pretreatment of JB6 cells with chlorogenic acid blocked UVB- or TPA-induced transactivation of AP-1 and NF-kappaB over the same dose range. At low concentrations, chlorogenic acid decreased the phosphorylation of c-Jun NH2-terminal kinases, p38 kinase, and MAPK kinase 4 induced by UVB/12-O-tetradecanoylphorbol-13-acetate, yet higher doses were required to inhibit extracellular signal-regulated kinases. Chlorogenic acid also increased the enzymatic activities of glutathione S-transferases (GST) and NAD(P)H: quinone oxidoreductase. Further studies indicated that chlorogenic acid could stimulate the nuclear translocation of Nrf2 (NF-E2-related factor) as well as subsequent induction of GSTA1 antioxidant response element (ARE)-mediated GST activity. The phosphatidylinositol 3-kinase pathway might be involved in the activation of Nrf2 translocation. These results provide the first evidence that chlorogenic acid could protect against environmental carcinogen-induced carcinogenesis and suggest that the chemopreventive effects of chlorogenic acid may be through its up-regulation of cellular antioxidant enzymes and suppression of ROS-mediated NF-kappaB, AP-1, and MAPK activation.

Topics: Agar; Animals; Antineoplastic Agents; Antioxidants; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Chlorogenic Acid; Cytosol; DNA-Binding Proteins; Dose-Response Relationship, Drug; Electric Impedance; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Genes, Reporter; Glutathione Transferase; Humans; Immunohistochemistry; Isoenzymes; Luciferases; MAP Kinase Signaling System; Mice; NADH, NADPH Oxidoreductases; NF-E2-Related Factor 2; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Plasmids; Protein Transport; Tetradecanoylphorbol Acetate; Trans-Activators; Transcription Factor AP-1; Transcriptional Activation; Ultraviolet Rays

Although nicotine has been suggested to promote lung carcinogenesis, the mechanism of its action in this process remains unknown. The present investigation demonstrates that the treatment of rat lung epithelial cells with nicotine for various periods differentially mobilizes multiple intracellular pathways. Protein kinase C and phosphoinositide 3-OH-kinase are transiently activated after the treatment. Also, Ras and its downstream effector ERK1/2 are activated after long term exposure to nicotine. The activation of Ras by nicotine treatment is responsible for the subsequent perturbation of the methotrexate (MTX)-mediated G1 cell cycle restriction as well as an increase in production of reactive oxygen species. When p53 expression is suppressed by introducing E6, persistent exposure to nicotine enables dihydrofolate reductase gene amplification in the presence of methotrexate (MTX) and the formation of the MTX-resistant colonies. Altering the activity of phosphoinositide 3-OH-kinase has no effect on dihydrofolate reductase amplification. However, the suppression of protein kinase C dramatically affects the colony formation in soft agar. Thus, our data suggest that persistent exposure to nicotine perturbs the G1 checkpoint and causes DNA damage through the increase of the production of reactive oxygen species. However, a third element rendered by loss of p53 is required for the initiation of the process of gene amplification. Under p53-deficient conditions, the establishment of a full oncogenic transformation, in response to long term nicotine exposure, is achieved through the cooperation of multiple signaling pathways.

Topics: Agar; Animals; Blotting, Southern; Cell Transformation, Neoplastic; Cells, Cultured; Cyclin D1; DNA Damage; Drug Resistance; Enzyme Activation; Epithelial Cells; Flow Cytometry; G1 Phase; Ganglionic Stimulants; Hydrogen Peroxide; Immunoblotting; Lung; Methotrexate; Nicotine; Phosphatidylinositol 3-Kinases; Promoter Regions, Genetic; Protein Kinase C; ras Proteins; Rats; Reactive Oxygen Species; Signal Transduction; Tetrahydrofolate Dehydrogenase; Thymidine; Time Factors; Tumor Suppressor Protein p53

Nijmegen breakage syndrome (NBS) is a chromosomal instability syndrome associated with cancer predisposition, radiosensitivity, microcephaly, and growth retardation. The NBS gene product, NBS1 (p95) or nibrin, is a part of the hMre11 complex, a central player associated with double strand break repair. We previously demonstrated that c-Myc directly activates NBS1 expression. Here we have shown that constitutive expression of NBS1 in Rat1a and HeLa cells induces/enhances their transformation. Repression of endogenous NBS1 levels using short interference RNA reduces the transformation activity of two tumor cell lines. Increased NBS1 expression is observed in 40-52% of non-small cell lung carcinoma, hepatoma, and esophageal cancer samples. NBS1 overexpression stimulates phosphatidylinositol (PI) 3-kinase activity, leading to increased phosphorylation levels of Akt and its downstream targets such as glycogen synthase kinase 3beta and mammalian target of rapamycin in different cell lines and tumor samples. Transformation induced by NBS1 overexpression can be inhibited by a PI3-kinase inhibitor (LY294002). Repression of endogenous Akt expression by short interference RNA decreases the transformation activity of Rat1a cells overexpressing NBS1. These results indicate that overexpression of NBS1 is an oncogenic event that contributes to transformation through the activation of PI3-kinase/Akt.

Topics: Agar; Animals; Blotting, Western; Cell Cycle Proteins; Cell Line, Tumor; Cell Transformation, Neoplastic; Chromones; Chromosomes; DNA Repair Enzymes; DNA-Binding Proteins; Enzyme Activation; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; HeLa Cells; Humans; Immunohistochemistry; Mice; Mice, Nude; Morpholines; MRE11 Homologue Protein; Neoplasm Transplantation; Nuclear Proteins; Phosphatidylinositol 3-Kinases; Phosphorylation; Plasmids; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-myc; Rats; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Sirolimus

Upon cell adhesion to extracellular matrix proteins, focal adhesion kinase (FAK) rapidly undergoes autophosphorylation on its Tyr-397 which consequently serves as a binding site for the Src homology 2 domains of the Src family protein kinases and several other intracellular signaling molecules. In this study, we have attempted to examine the effect of the FAK Y397F mutant on v-Src-stimulated cell transformation by establishing an inducible expression of the Y397F mutant in v-Src-transformed FAK-null (FAK(-/-)) mouse embryo fibroblasts. We found that the FAK Y397F mutant had both positive and negative effects on v-Src-stimulated cell transformation; it promoted v-Src-stimulated invasion, but on the other hand it inhibited the v-Src-stimulated anchorage-independent cell growth in vitro and tumor formation in vivo . The positive effect of the Y397F mutant on v-Src-stimulated invasion was correlated with an increased expression of matrix metalloproteinase-2, both of which were inhibited by the specific phosphatidylinositol 3-kinase inhibitor wortmannin or a dominant negative mutant of AKT, suggesting a critical role for the phosphatidylinositol 3-kinase/AKT pathway in both events. However, the expression of the Y397F mutant rendered v-Src-transformed FAK(-/-) cells susceptible to anoikis, correlated with suppression on v-Src-stimulated activation of ERK and AKT. In addition, under anoikis stress, the induction of the Y397F mutant in v-Src-transformed FAK(-/-) cells selectively led to a decrease in the level of p130(Cas), but not other focal adhesion proteins such as talin, vinculin, and paxillin. These results suggest that FAK may increase the susceptibility of v-Src-transformed cells to anoikis by modulating the level of p130(Cas).

Topics: Actins; Agar; Androstadienes; Animals; Anoikis; Binding Sites; Cell Adhesion; Cell Cycle; Cell Line; Cell Movement; Cell Transformation, Neoplastic; Collagen; Crk-Associated Substrate Protein; Drug Combinations; Enzyme Activation; Enzyme Inhibitors; Fibroblasts; Focal Adhesion Protein-Tyrosine Kinases; Focal Adhesions; Gelatin; Genes, Dominant; Immunoblotting; Immunoprecipitation; Laminin; Matrix Metalloproteinase 2; Mice; Mice, Nude; Mice, Transgenic; Microscopy, Fluorescence; Mutation; Neoplasm Invasiveness; Oncogene Protein pp60(v-src); Paxillin; Phosphatidylinositol 3-Kinases; Phosphorylation; Proteoglycans; Proto-Oncogene Proteins c-akt; Signal Transduction; src-Family Kinases; Talin; Time Factors; Transfection; Tyrosine; Vinculin; Wortmannin

PAK4 is a member of the group B family of p21-activated kinases. Its expression is elevated in many cancer cell lines, and activated PAK4 is highly transforming, suggesting that it plays an important role in tumorigenesis. Although most previous work was carried out with overexpressed PAK4, here we used RNA interference to knock down endogenous PAK4 in cancer cells. By studying PAK4 knockdown HeLa cells, we demonstrated that endogenous PAK4 is required for anchorage-independent growth. Because cell survival is a key part of tumorigenesis and anchorage-independent growth, we studied whether PAK4 has a role in protecting cells from cell death. To address this, we studied the role for PAK4 downstream to the tumor necrosis factor (TNF) alpha receptor. Although overexpressed PAK4 was previously shown to abrogate proapoptotic pathways, here we demonstrate that endogenous PAK4 is required for the full activation of prosurvival pathways induced by TNFalpha. Our results indicate that PAK4 is required for optimal binding of the scaffold protein TRADD to the activated TNFalpha receptor through both kinase-dependent and kinase-independent mechanisms. Consequently, activation of several prosurvival pathways, including the NFkappaB and ERK pathways, is reduced in the absence of PAK4. Interestingly, constitutive activation of the NFkappaB and ERK pathways could compensate for the lack of PAK4, indicating that these pathways function downstream to PAK4. The role for PAK4 in regulating prosurvival pathways is a completely new function for this protein, and the connection between PAK4 and cell survival under stress helps explain its role in tumorigenesis and development.

Topics: Agar; Apoptosis; Blotting, Western; Cell Death; Cell Line, Transformed; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cell Transformation, Neoplastic; Flow Cytometry; Genetic Vectors; HeLa Cells; Humans; Immunoprecipitation; NF-kappa B; p21-Activated Kinases; Protein Binding; Protein Serine-Threonine Kinases; Protein Structure, Tertiary; Receptors, Tumor Necrosis Factor; RNA Interference; RNA, Small Interfering; Time Factors; Transfection; Tumor Necrosis Factor Receptor-Associated Peptides and Proteins; Tumor Necrosis Factor-alpha; Ultraviolet Rays

Etk, the 70-kDa member of the Tec family of nonreceptor protein tyrosine kinases, is expressed in a variety of hematopoietic, epithelial, and endothelial cells and was shown to be involved in several cellular processes, including proliferation, differentiation, and motility. In this study, we describe a novel approach using a human single-domain antibody phage display library for the generation of intrabodies directed against Etk. These single-domain antibodies bind specifically to recombinant Etk and efficiently block its kinase activity. When expressed in transformed cells, these antibodies associated tightly with Etk, leading to significant blockade of Etk enzymatic activity and inhibition of clonogenic cell growth in soft agar. Our results indicate that Etk may play a role in Src-induced cellular transformation and thus may represent a good target for cancer intervention. Furthermore, our single-domain antibody-based intrabody system proves to be an excellent tool for future intracellular targeting of other signaling molecules.

Topics: Agar; Animals; Antineoplastic Agents; Blotting, Western; Cell Differentiation; Cell Proliferation; Cell Transformation, Neoplastic; Cloning, Molecular; DNA; Dose-Response Relationship, Drug; Enzyme Activation; Glutathione Transferase; Humans; Immunoprecipitation; Mice; NIH 3T3 Cells; Peptide Library; Phosphorylation; Protein Binding; Protein Structure, Tertiary; Protein-Tyrosine Kinases; Recombinant Proteins; RNA Interference; Signal Transduction; Transfection

Receptor tyrosine kinases are integral components of cellular signaling pathways and are frequently deregulated in malignancies. The NTRK family of neurotrophin receptors mediate neuronal cell survival and differentiation, but altered NTRK signaling has also been implicated in oncogenesis. The ETV6-NTRK3 (EN) gene fusion occurs in human pediatric spindle cell sarcomas and secretory breast carcinoma, and encodes the oligomerization domain of the ETV6 transcription factor fused to the protein-tyrosine kinase domain of NTRK3. The EN protein functions as a constitutively active protein-tyrosine kinase with potent transforming activity in multiple cell lineages, and EN constitutively activates both the Ras-MAPK and phosphatidylinositol 3-kinase-Akt pathways. EN transformation is associated with constitutive tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1). Further, IRS-1 functions as the adaptor protein linking EN to downstream signaling pathways. However, the exact nature of the EN-IRS-1 interaction remains unknown. We now demonstrate that EN specifically binds the phosphotyrosine binding domain of IRS-1 via an interaction at the C terminus of EN. An EN mutant lacking the C-terminal 19 amino acids does not bind IRS-1 and lacks transforming ability. Moreover, expression of an IRS-1 polypeptide containing the phosphotyrosine binding domain acts in a dominant negative manner to inhibit EN transformation, and overexpression of IRS-1 potentiates EN transforming activity. These findings indicate that EN.IRS-1 complex formation through the NTRK3 C terminus is essential for EN transformation.

Topics: Agar; Amino Acid Sequence; Animals; Binding Sites; Cell Differentiation; Cell Line; Cell Line, Tumor; Cell Survival; Cell Transformation, Neoplastic; Conserved Sequence; DNA-Binding Proteins; DNA, Complementary; Enzyme Activation; ETS Translocation Variant 6 Protein; Fibroblasts; Genes, Dominant; Genetic Vectors; Humans; Insulin Receptor Substrate Proteins; Mice; Mice, Nude; Molecular Sequence Data; Mutagenesis, Site-Directed; Neurons; NIH 3T3 Cells; Oncogene Proteins, Fusion; Phosphatidylinositol 3-Kinases; Phosphoproteins; Phosphotyrosine; Protein Binding; Protein Structure, Tertiary; Proto-Oncogene Proteins c-ets; Receptor, trkC; Repressor Proteins; Retroviridae; Sequence Homology, Amino Acid; Signal Transduction; Time Factors; Tyrosine

Histone acetylase and histone deacetylase are two crucial enzymes that determine the structure of chromatin, regulating gene expression. In this study, we observed that trichostatin A (TSA), a specific histone deacetylase inhibitor, could effectively inhibit the growth of v-Src-transformed (IV5) cells and abrogate their ability to form colonies in soft agar. Further analysis demonstrated that, although TSA reduced the expression of Eps8 in a dose- and time-dependent manner, both the protein expression and kinase activity of v-Src remained constant, and the abundance and phosphotyrosine levels of Src substrates, including cortactin, focal adhesion kinase, p130(Cas), paxillin, and Shc, were not altered. Notably, removal of TSA from the medium restored not only the expression of Eps8, but also cellular growth. Northern and reverse transcription-PCR analyses revealed the significant reduction of eps8 transcripts in TSA-treated IV5 cells relative to control cells. When active Src-expressing chicken embryonic cells were forced to overexpress p97(Eps8), they became resistant to TSA-mediated anti-proliferation. Furthermore, using small interference RNA of eps8, we demonstrated the requirement for Eps8 in IV5 cell proliferation. Thus, our results highlight a critical role for p97(Eps8) in TSA-exerted growth inhibition of v-Src-transformed cells.

Topics: Adaptor Proteins, Signal Transducing; Adaptor Proteins, Vesicular Transport; Agar; Animals; Blotting, Northern; Cell Division; Cell Line; Cell Transformation, Neoplastic; Chickens; Cortactin; Cytoskeletal Proteins; Dose-Response Relationship, Drug; Down-Regulation; Enzyme Inhibitors; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Histones; Hydroxamic Acids; Immunoblotting; Microfilament Proteins; Mutation; Paxillin; Phosphoproteins; Phosphotyrosine; Precipitin Tests; Protein-Tyrosine Kinases; Proteins; Rats; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Shc Signaling Adaptor Proteins; Src Homology 2 Domain-Containing, Transforming Protein 1; src-Family Kinases; Time Factors; Transfection

The c-myc proto-oncogene encodes a ubiquitous transcription factor involved in the control of cell growth and differentiation and implicated in inducing tumorigenesis. Understanding the function of c-Myc and its role in cancer depends upon the identification of c-Myc target genes. Heat shock protein 90 (HSP90) is involved in the folding of proteins such as signal transduction molecules (Src, Raf1, cdk4) and steroid receptors and in enhancing the activity of telomerase and nitric-oxide synthase. Here we show that c-Myc directly activates HSP90A transcription. c-Myc-mediated induction of HSP90A transcription occurs in different tissues, is independent of cell proliferation, and is mediated by a c-Myc binding site in the proximal promoter region of HSP90A gene. Overexpression of HSP90A in Rat1a cells induces transformation. Short interference RNA of HSP90A/Hsp86alpha reduces transformation activity in HeLa and RatMyc cells. These results indicate that by induction of HSP90A c-Myc may control the activity of multiple signal pathways involved in cellular transformation.

Topics: Agar; Animals; Binding Sites; Blotting, Northern; Blotting, Western; Cell Division; Cell Line; Cell Line, Tumor; Cell Transformation, Neoplastic; Chromatin; Cloning, Molecular; Genes, Reporter; HeLa Cells; HSP90 Heat-Shock Proteins; Humans; Luciferases; Mice; Mice, Nude; NIH 3T3 Cells; Plasmids; Precipitin Tests; Promoter Regions, Genetic; Proto-Oncogene Mas; Proto-Oncogene Proteins c-myc; Rats; RNA, Small Interfering; Signal Transduction; Transcription, Genetic; Transfection; U937 Cells; Up-Regulation

To investigate the multistage process of carcinogenesis, the progressive alteration of the morphology, telomerase, cytogenesis, oncogenes and tumorigenicity in the process of immortalization and malignant transformation of the human fetal esophageal epithelial cell (SHEE) was studied. The SHEE cells were immortalized by gene E6E7 of human papilloma virus (HPV) type 18 in our laboratory and continually cultivated over 100 passages, which had been malignantly transformed. Cells at the 11th, 35th, 65th and 100th passage were examined according to the following criteria: morphological changes of cell growth, contact-inhibition and anchorage-independent growth (AIG); the cell proliferative and apoptotic index; the modal number of chromosomes; c-myc, p53, bcl-2, ras; telomere length and activities of telomerase and tumorigenicity in nude mice or severe combined immunodeficient (SCID) mice. The cells of the 11th passage were well differentiated and the cells of 100th passage were relatively poorly differentiated with polymorphism, while the cells of 35th and 65th had two distinct differentiations. The proliferative indexes were 21.1%, 32.5%, 33.2%, and 40.9% and the apoptotic indexes were 3.3%, 2.7%, 3.5%, 2.7% in the 11th, 35th, 65th and 100th passage respectively. Karyotypes of four cell passages belonged to hyperdiploidy and hypotriploidy. C-myc, ras, p53 genes were low in the 10th and 35th, and high in the 65th and 100th passage, but bcl-2 was low in 4 passages. Telomere length sharply decreased from normal fetal esophagus cells until the 35th passage, but it was stably expressed in the 65th and 100th passage. The activities of telomerase were expressed in cells of the 35th, 65th and 100th passages. The efficiency of AIG varied in different passages of the SHEE cell and was absent in the 11th passage, low efficiency in the 35th passage and 65th passage, and high efficiency in the 100th passage. Transplanted cells of the 65th and 100th passage into SCID mice resulted in tumor formation, but only the 100th passage cells could grow in nude mice. All of these characteristic changes were in dynamic progressive process. These data demonstrate that carcinogenesis of esophageal epithelial cells induced by HPV is the multistage process, which goes through the initial, immortal, premalignant and malignant transformation stages. The generation of esophageal carcinoma is caused by the accumulation of cellular, genetic and molecular changes.

Topics: Agar; Animals; Apoptosis; Blotting, Western; Cell Cycle; Cell Division; Cell Line; Cell Transformation, Neoplastic; Epithelial Cells; Esophageal Neoplasms; Humans; Karyotyping; Mice; Mice, Inbred BALB C; Mice, Nude; Mice, SCID; Microscopy, Phase-Contrast; Neoplasm Transplantation; Papillomaviridae; Ploidies; Polymerase Chain Reaction; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-myc; RNA, Messenger; Telomerase; Telomere; Time Factors

Arsenic and cadmium (Cd(+2)) are human carcinogens, and epidemiological studies have implicated both pollutants in the development of urinary bladder cancer. Despite this epidemiological base, it is unknown if either Cd(+2) or arsenite (As(+3)) can directly cause the malignant transformation of human urothelial cells. The goal of this study was to determine if Cd(+2) and/or As(+3) are able to cause the malignant transformation of human urothelial cells. The strategy employed was to expose the nontumorigenic urothelial cell line UROtsa to long-term in vitro exposure to Cd(+2) and As(+3), with the endpoint being the ability of the cells to form colonies in soft agar and tumors when heterotransplanted into nude mice. It was demonstrated that a long-term exposure to either 1 M Cd(+2) or 1 M As(+3) resulted in the selection of cells that were able to form colonies in soft agar and tumors when heterotransplanted into nude mice. The histology of the tumor heterotransplants produced by UROtsa cells malignantly transformed by Cd(+2) had epithelial features consistent with those of a classic transitional-cell carcinoma of the bladder. The histology of the tumor heterotransplants produced by cells malignantly transformed by As(+3) was unique in that the cells displayed a prominent squamoid differentiation.

Topics: Agar; Animals; Arsenicals; Cadmium Compounds; Cell Culture Techniques; Cell Division; Cell Line; Cell Transformation, Neoplastic; Dose-Response Relationship, Drug; Humans; Mice; Mice, Nude; Neoplasm Transplantation; Neoplasms, Experimental; Time Factors; Urinary Bladder; Urothelium

Serum- and glucocorticoid-regulated kinase (SGK) is a serine kinase that has a catalytic domain homologous to that of Akt, but lacks the pleckstrin homology domain present in Akt. Akt reportedly plays a key role in various cellular actions, including glucose transport, glycogen synthesis, DNA synthesis, anti-apoptotic activity, and cell proliferation. In this study, we attempted to reveal the different roles of SGK and Akt by overexpressing active mutants of Akt and SGK. We found that adenovirus-mediated overexpression of myristoylated (myr-) forms of Akt resulted in high glucose transport activity in 3T3-L1 adipocytes, phosphorylated glycogen synthase kinase-3 (GSK3) and enhanced glycogen synthase activity in hepatocytes, and the promotion of DNA synthesis in interleukin-3-dependent 32D cells. In addition, stable transfection of myr-Akt in NIH3T3 cells induced an oncogenic transformation in soft agar assays. The active mutant of SGK (D-SGK, substitution of Ser422 with Asp) and myr-SGK were shown to phosphorylate GSK3 and to enhance glycogen synthase activity in hepatocytes in a manner very similar to that observed for myr-Akt. However, despite the comparable degree of GSK3 phosphorylation between myr-Akt and d-SGK or myr-SGK, d-SGK and myr-SGK failed to enhance glucose transport activity in 3T3-L1 cells, DNA synthesis in 32D cells, and oncogenic transformation in NIH3T3 cells. Therefore, the different roles of SGK and Akt cannot be attributed to ability or inability to translocate to the membrane thorough the pleckstrin homology domain, but rather must be attributable to differences in the relatively narrow substrate specificities of these kinases. In addition, our observations strongly suggest that phosphorylation of GSK3 is either not involved in or not sufficient for GLUT4 translocation, DNA synthesis, or oncogenic transformation. Thus, the identification of substrates selectively phosphorylated by Akt, but by not SGK, may provide clues to clarifying the pathway leading from Akt activation to these cellular activities.

Topics: 3T3 Cells; Adenoviridae; Adipocytes; Agar; Animals; Biological Transport; Blotting, Western; Catalytic Domain; Cell Division; Cell Line; Cell Transformation, Neoplastic; DNA; Fibroblasts; Gene Transfer Techniques; Glucose; Glucose Transporter Type 4; Glycogen Synthase; Hepatocytes; Immediate-Early Proteins; Immunoblotting; Interleukin-3; Mice; Monosaccharide Transport Proteins; Muscle Proteins; Mutation; Neoplasms; Nuclear Proteins; Phosphorylation; Precipitin Tests; Protein Serine-Threonine Kinases; Protein Structure, Tertiary; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Rats; Time Factors; Transfection

Recent studies have demonstrated that introduction of hTERT in combination with SV40 large T antigen (LT), small t antigen (st), and H-rasV12 suffices to transform many primary human cells. In human mammary epithelial cells (HMECs) expressing elevated c-Myc, activated H-Ras is dispensable for anchorage-independent growth. Using this system, we show that st activates the PI3K pathway and that constitutive PI3K signaling substitutes for st in transformation. Moreover, using constitutively active versions of Akt1 and Rac1, we show that these downstream pathways of PI3K synergize to achieve anchorage-independent growth. At lower levels of c-myc expression, activated PI3K also replaces st to complement H-rasV12 and LT and confers both soft agar growth and tumorigenicity. However, elevated c-myc expression cannot replace H-rasV12 for tumorigenesis. These observations begin to define the pathways perturbed during the transformation of HMECs.

Topics: Agar; Alternative Splicing; Cell Division; Cell Transformation, Neoplastic; DNA, Complementary; Dose-Response Relationship, Drug; Enzyme Activation; Fibroblasts; Genetic Vectors; Humans; Immunoblotting; Mammary Glands, Human; Phosphatidylinositol 3-Kinases; Phosphorylation; Precipitin Tests; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-myc; rac1 GTP-Binding Protein; Retroviridae; Time Factors; Transfection

Several reports have shown that the ectopic expression of the human telomerase catalytic subunit gene (hTERT) leads to an indefinite extension of the life span of human fibroblasts cultured in vitro without the appearance of cancer-associated changes. We infected two fibroblast strains derived from centenarian individuals with an hTERT containing retrovirus and isolated transduced massive populations (cen2tel and cen3tel). In both populations, hTERT expression reconstituted telomerase activity and extended the life span. In cen2tel, a net telomere lengthening was observed while, in cen3tel, telomeres stabilized at a length lower than that detected in senescent parental cells. Interestingly, both cen2tel and cen3tel cells developed chromosome anomalies, numerical first and structural thereafter. Moreover, cen3tel cells acquired the ability to grow in the absence of solid support, a typical feature of transformed cells. The results we present here highlight an unexpected possible outcome of cellular immortalization driven by telomerase reactivation, and indicate that, in some cases, an artificial extension of cellular replicative capacity can increase the probability of occurrence of genomic alterations, which can lead to cellular transformation.

Topics: Agar; Aged; Aged, 80 and over; Aging; Cell Adhesion; Cell Division; Cell Transformation, Neoplastic; Cellular Senescence; Centromere; Chromosome Aberrations; DNA; DNA-Binding Proteins; Female; Fibroblasts; Humans; Karyotyping; Retroviridae; Telomerase; Telomere; Time Factors

A major prognostic marker for neuroblastoma (Nb) is N-myc gene amplification, which predicts a poor clinical outcome. We sought genes differentially expressed on a consistent basis between multiple human Nb cell lines bearing normal versus amplified N-myc, in hopes of finding target genes that might clarify how N-myc overexpression translates into poor clinical prognosis. Using differential display, we find the previously described growth-inhibitory gene Ndrg1 is strongly repressed in all tested Nb cell lines bearing N-myc amplification, as well as in a neuroepithelioma line with amplified c-myc. Overexpression of N-myc in non-amplified Nb cells leads to repression of Ndrg1, as does activation of an inducible c-myc transgene in fibroblasts. Conversely, N-myc downregulation in N-myc-amplified Nb cells results in re-expression of the Ndrg1, and stimuli known to induce Ndrg1 do so in Nb cells while simultaneously down-regulating N-myc. Relevant to these results, we demonstrate an in vitro interaction of Myc protein with the Ndrg1 core promoter. We also find that Ndrg1 levels increase dramatically during in vitro differentiation of two cell lines modeling neural and glial development, while c- and N-myc levels decline. Our results combined with previous information on the Ndrg1 gene product suggest that downregulation of this gene is an important component of N-Myc effects in neuroblastomas with poor clinical outcome. In support of this notion, we find that re-expression of Ndrg1 in high-Myc Nb cells results in smaller cells with reduced colony size in soft-agar assays, further underscoring the functional significance of this gene in human neuroblastoma cells.

Topics: Agar; Biomarkers, Tumor; Blotting, Northern; Cell Cycle Proteins; Cell Differentiation; Cell Division; Cell Line, Tumor; Cell Transformation, Neoplastic; Cloning, Molecular; Down-Regulation; Gene Expression Profiling; Glutathione Transferase; Humans; Immunoblotting; Intracellular Signaling Peptides and Proteins; Neuroblastoma; Neuroectodermal Tumors, Primitive, Peripheral; Prognosis; Promoter Regions, Genetic; Protein Binding; Proto-Oncogene Proteins c-myc; Reverse Transcriptase Polymerase Chain Reaction; Ribonucleases; RNA, Messenger; Sequence Analysis, DNA; Time Factors

Mutations within members of the EGF/ErbB receptor family frequently release the oncogenic potential of these receptors, resulting in the activation of downstream signaling events independent of ligand regulatory constraints. We previously have demonstrated that the signal transduction events originating from S3-v-ErbB, a ligand-independent, oncogenic EGF receptor mutant, are qualitatively distinct from the ligand-dependent mitogenic signaling pathways associated with the wild-type EGF receptor. Specifically, expression of S3-v-ErbB in primary fibroblasts results in anchorage-independent growth, increased invasive potential, and the formation of a transformation-specific phosphoprotein signaling complex, all in a Ras-independent manner. Here we demonstrate the transformation-specific interaction between two components of this complex: the adaptor protein Grb2 and the cytoskeletal regulatory protein caldesmon. This interaction is mediated via both the amino-terminal SH3 and central SH2 domains of Grb2, and the amino-terminal (myosin-binding) domain of caldesmon. Expression of a dominant-negative Grb2 deletion mutant, which lacks the carboxy-terminal SH3 domain, in fibroblasts expressing S3-v-ErbB results in a reduction in phosphoprotein complex formation, the loss of anchorage-independent growth, and a reduction in invasive potential. Together, these results demonstrate a Ras-independent role for Grb2 in modulating cytoskeletal function during ligand-independent EGF receptor-mediated transformation, and provide further support for the hypothesis that ligand-independent oncogenic signaling is qualitatively distinct from ligand-dependent mitogenic signaling by the EGF receptor.

Topics: Actins; Adaptor Proteins, Signal Transducing; Agar; Animals; Calmodulin-Binding Proteins; Cell Line, Transformed; Cell Transformation, Neoplastic; Cells, Cultured; Chick Embryo; Cytoskeleton; ErbB Receptors; Fibroblasts; Glutathione Transferase; GRB2 Adaptor Protein; Ligands; Microscopy, Fluorescence; Models, Biological; Mutation; Neoplasm Invasiveness; Phosphorylation; Phosphotyrosine; Precipitin Tests; Protein Structure, Tertiary; Proteins; Recombinant Fusion Proteins; Retroviridae; Signal Transduction; src Homology Domains; Tyrosine

Infection with human herpes virus 8 (HHV8) has been associated with Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. HHV8 encodes for a viral FLICE-inhibitory protein (vFLIP), designated K13, which resembles the prodomain of caspase-8 in structure and has been shown to protect cells against death receptor-induced apoptosis in vitro and in vivo. In this report, we present evidence that HHV8 vFLIP also possesses the unique ability of transforming Rat-1 and Balb/3T3 fibroblast cells, which is not shared by other vFLIPs. Rat-1 cells expressing HHV8 vFLIP form colonies in soft agar and form tumors in nude mice. The transforming ability of HHV8 vFLIP is associated with the activation of the NF-kappaB pathway and is blocked by molecular and chemical inhibitors of this pathway. Our results suggest that vFLIP K13 has activity beyond its role as an inhibitor of death receptor signaling and may play a causative role in the pathogenesis of HHV8-associated malignancies. Furthermore, inhibitors of the NF-kappaB pathway may have a role in the treatment of malignancies linked to HHV8 infection.

Topics: Adenoviridae; Agar; Amino Acid Sequence; Animals; Apoptosis; Blotting, Southern; Blotting, Western; Caspase 8; Caspases; Cell Death; Cell Division; Cell Line; Cell Transformation, Neoplastic; Genetic Vectors; Herpesvirus 8, Human; Humans; Luciferases; Mice; Mice, Inbred BALB C; Mice, Nude; Microscopy, Phase-Contrast; Molecular Sequence Data; NF-kappa B; Rats; Retroviridae; Sequence Homology, Amino Acid; Signal Transduction; Time Factors; Viral Proteins

Overexpression of the translation initiation factor eIF4E results in transformation of normal fibroblasts as a single-hit oncogene. This implies that eIF4E must affect several pathways leading to transformation. The oncogenic potential of eIF4E is probably realized by elevating the translational efficiency of some oncogene and growth-promotion transcripts that are normally repressed by their 5'UTR (untranslated region). To address this possibility, we have cloned mRNAs whose polysomal representation increases upon overexpression of eIF4E. Among these mRNAs, we now report the isolation of a clone corresponding to the src-like kinase yes. The yes mRNA contains a long 5'UTR with characteristic features of a typical translationally repressed transcript. This was confirmed by analysis of the distribution of yes mRNA after sedimentation in sucrose gradients. Increased utilization of yes mRNA resulted in elevated expression of the protein product in cells transformed with eIF4E, and suggested that overexpression of Yes could contribute to eIF4E-mediated transformation. To test this, we monitored the malignant properties of MM3MG-4E cells after treatment with PP2, a specific inhibitor of src kinases. Growth in soft agar and saturation densities were significantly reduced after treatment with PP2, but treatment of mice harboring MM3MG-4E tumors with PP2 did not affect tumor growth. However, transformation of yes-null fibroblasts by eIF4E was significantly impaired.

Topics: 5' Untranslated Regions; Agar; Animals; Blotting, Northern; Cell Line, Tumor; Cell Transformation, Neoplastic; Centrifugation, Density Gradient; CHO Cells; Cloning, Molecular; Cricetinae; Eukaryotic Initiation Factor-4E; Fibroblasts; Mice; Mice, Nude; Polyribosomes; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-yes; RNA; RNA, Messenger; Seminal Plasma Proteins; Time Factors; Transcription, Genetic; Up-Regulation

Insulin-like growth factor receptor 1 (IGFR1) plays a crucial role in oncogenic transformation [C. Sell et al., Mol. Cell. Biol., 14: 3604-3612,1994]. Compared with the normal human mammary epithelial cell line MCF12A, MCF7 human mammary carcinoma cells overexpress IGFR1 on the cell surface. To measure the effects of IGFR1 inhibition on tumor cells, we tested two mouse neutralizing antibodies against human IGFR1 in cell-based assays. Both MAB391 and anti-IR3 antibodies inhibit IGFR1 autophosphorylation upon IGF-I ligand stimulation with IC50s of 0.58 and 0.80 nM, respectively. When cells were treated with neutralizing anti-IGFR1 antibodies for > or = 4 h, the total receptor level was dramatically decreased. IGF-I-stimulated activation of AKT was also inhibited by anti-IGFR1 antibodies. Furthermore, MAB391 and anti-IR3 inhibited the growth of MCF7 cells in soft agar. In addition to MCF7 cells, MAB391 also inhibited IGFR1 autophosphorylation and induced IGFR1 down-modulation in HT29 colorectal and Du145 prostate cancer cells. Therefore, neutralizing antibodies against IGFR1 represent a valid approach to inhibit growth of tumor cells.

Topics: Agar; Antibodies, Monoclonal; Blotting, Western; Cell Transformation, Neoplastic; Dose-Response Relationship, Drug; Down-Regulation; Electrophoresis, Polyacrylamide Gel; Humans; Inhibitory Concentration 50; Insulin-Like Growth Factor I; Lysosomes; Phosphorylation; Precipitin Tests; Receptor, IGF Type 1; Signal Transduction; Time Factors; Tumor Cells, Cultured

Elevated hyaluronan biosynthesis and matrix deposition correlates with cell proliferation and migration. We ectopically expressed three isoforms of hyaluronan synthase (HAS1, HAS2, or HAS3) in nontransformed rat 3Y1 cells and observed a de novo, massive formation of a hyaluronan matrix that resulted in a partial loss of contact-mediated inhibition of cell growth and migration. All three HAS transfectants showed an enhanced motility in scratch wound assays, and a significant increase in their confluent cell densities. In high-density cultures, the HAS transfectants had a fibroblastic cell shape and markedly formed overlapping cell layers. This phenotype was more pronounced in the HAS2 transfectants than HAS1 or HAS3 transfectants, and occurred with significant alterations in the microfilament organization and N-cadherin distribution at the cell-cell border. Inhibition of a phosphatidylinositol 3-kinase (PI3-kinase) pathway resulted in reacquisition of the normal phenotype of HAS2 transfectants, suggesting that the intracellular PI3-kinase signaling regulates diminution of contact inhibition induced by formation of the massive hyaluronan matrix. Our observations suggest that hyaluronan and its matrix can modulate contact inhibition of cell growth and migration, and provide evidence for functional differences between hyaluronan synthesized by the different HAS proteins.

Topics: Agar; Animals; Cell Count; Cell Division; Cell Line; Cell Movement; Cell Size; Cell Transformation, Neoplastic; Contact Inhibition; Cytoskeleton; Erythrocytes; Extracellular Matrix; Fibroblasts; Flow Cytometry; Gene Expression; Glucuronosyltransferase; Glycosyltransferases; Hyaluronan Synthases; Hyaluronic Acid; Isoenzymes; Membrane Proteins; Microscopy, Electron; Phenotype; Phosphatidylinositol 3-Kinases; Rats; Sheep; Transfection; Transferases; Wound Healing; Xenopus Proteins

p204 overexpression in retinoblastoma (Rb)-/- mouse embryo fibroblasts or transfection of p204 mutated at both Rb-binding sites confer growth advantages, resulting in a significantly higher number of foci in a cell focus assay. To investigate the possibility that mutated p204 acquires malignant transformation capability, NIH3T3 cells were stably transfected with the expression vector pRcRSV204 double-mutant (p204dm) harboring both the C-terminal deletion up to amino acid 568 and the point mutation from glutamic acid to lysine at position 427, and analyzed for markers typical of cell immortalization and transformation. We detected a greater abundance of cell colonies in soft agar with p204dm-expressing cells than vector control cells. The p204dm-transfected cells also displayed two other characteristics associated with malignant transformation, i.e. growth under low-serum conditions and formation of tumors in athymic nude mice. Moreover, their telomerase activity was significantly higher than in the vector control cells. It would thus seem that p204, devoid of functional Rb-binding motifs, can become oncogenic.

Topics: 3T3 Cells; Agar; Animals; Binding Sites; Cell Division; Cell Transformation, Neoplastic; Fibroblasts; Mice; Mice, Nude; Neoplasm Transplantation; Neoplastic Stem Cells; Nuclear Proteins; Phosphoproteins; Point Mutation; Retinoblastoma Protein; Sequence Deletion; Telomerase; Transfection

Most molecular biology and biochemical analyses use cultured cells grown in anchorage-dependent monolayer conditions. The standard oncogenic transformation assay for cell lines is usually performed in soft agar rather than in monolayers because of the higher transformation efficiency of cells in soft agar. However, cells suspended in soft agar cannot be readily recovered for studying inducible biochemical and molecular events. We developed an over-agar assay that enables us to study tumor promoter-induced cell transformation and the associated biochemical or molecular events under anchorage-independent conditions.

Topics: Agar; Animals; Cell Adhesion; Cell Count; Cell Separation; Cell Transformation, Neoplastic; Cells, Cultured; Culture Media; Epidermal Cells; Epidermis; Mice

Microcell-mediated chromosome transfer allows for the introduction of normal chromosomes into tumor cells in an effort to identify putative tumor suppressor genes. We have used this approach to introduce an intact copy of chromosome 18 into the prostate cancer cell line DU145, and independently to introduce human chromosomes 8 and 18 into the prostate cancer cell line TSU-PR1. Introduction of an extra copy of human chromosome 8 had no effect on the growth properties in vitro or the tumorigenicity in vivo of TSU-PR1 cells. However, microcell hybrids containing an introduced copy of human chromosome 18 exhibited a longer population doubling time, retarded growth in soft agar, and slowed tumor growth in athymic nude mice. These experiments provide functional evidence for the presence of one or more tumor suppressor genes on human chromosome 18 that are involved in prostate cancer.

Topics: Agar; Animals; Cell Culture Techniques; Cell Division; Cell Transformation, Neoplastic; Chromosomes, Human, Pair 18; Gene Transfer Techniques; Genes, Tumor Suppressor; Humans; Hybrid Cells; Male; Mice; Mice, Nude; Neoplasm Transplantation; Prostatic Neoplasms; Tumor Cells, Cultured

Topics: 3T3 Cells; Agar; Animals; Antigens, Polyomavirus Transforming; Cell Adhesion; Cell Communication; Cell Count; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Culture Media; Cytopathogenic Effect, Viral; Methylcellulose; Mice; Mice, Nude; Mutation; Neoplasm Transplantation; Polyhydroxyethyl Methacrylate; Rats; Simian virus 40; Virology

Skp2 is a member of the F-box family of substrate-recognition subunits of SCF ubiquitin-protein ligase complexes that has been implicated in the ubiquitin-mediated degradation of several key regulators of mammalian G(1) progression, including the cyclin-dependent kinase inhibitor p27, a dosage-dependent tumor suppressor protein. In this study, we examined Skp2 and p27 protein expression by immunohistochemistry in normal oral epithelium and in different stages of malignant oral cancer progression, including dysplasia and oral squamous cell carcinoma. We found that increased levels of Skp2 protein are associated with reduced p27 in a subset of oral epithelial dysplasias and carcinomas compared with normal epithelial controls. Tumors with high Skp2 (>20% positive cells) expression invariably showed reduced or absent p27 and tumors with high p27 (>20% positive cells) expression rarely showed Skp2 positivity. Increased Skp2 protein levels were not always correlated with increased cell proliferation (assayed by Ki-67 staining), suggesting that alterations of Skp2 may contribute to the malignant phenotype without affecting proliferation. Skp2 protein overexpression may lead to accelerated p27 proteolysis and contribute to malignant progression from dysplasia to oral epithelial carcinoma. Moreover, we also demonstrate that Skp2 has oncogenic potential by showing that Skp2 cooperates with H-Ras(G12V) to malignantly transform primary rodent fibroblasts as scored by colony formation in soft agar and tumor formation in nude mice. The observations that Skp2 can mediate transformation and is up-regulated during oral epithelial carcinogenesis support a role for Skp2 as a protooncogene in human tumors.

Topics: Agar; Animals; Carcinoma, Squamous Cell; Cell Cycle Proteins; Cell Transformation, Neoplastic; Cells, Cultured; Cyclin-Dependent Kinase Inhibitor p27; Disease Progression; Epithelial Cells; Epithelium; Fibroblasts; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Ki-67 Antigen; Microtubule-Associated Proteins; Mouth Neoplasms; Neoplasm Invasiveness; Oncogene Proteins; Proto-Oncogene Proteins p21(ras); Rats; S-Phase Kinase-Associated Proteins; Transfection; Tumor Suppressor Proteins

Int6/eIF3-p48 was first identified as a common integration site for MMTV in mouse mammary tumors. In all cases, the MMTV integration event resulted in an interruption of the normal Int6 transcript from one allele leaving the second allele intact and operative. We hypothesize that insertion of MMTV into Int6 results in a mutated allele that encodes a shortened Int6 mRNA and protein (Int6sh), which either modifies normal Int6 function or possesses a new independent function. To confirm the transforming potential of the mutation and its dominant function, we transfected two mammary epithelial cell lines, MCF10A (human), and HC11 (mouse), with Int6sh under the control of the elongation factor-1alpha (eEF1A) promoter. Expression of Int6sh in MCF10A and HC11 mammary epithelial cells leads to anchorage-independent growth in soft agar indicative of a transformed phenotype. Colonies selected from agar exhibited high levels of mutated Int6sh and wild type Int6 RNA transcripts by RT-PCR and Northern blot analysis. In addition, Int6sh transformed MCF10A and HC11 cells formed nodular growths, in vivo, in immune compromised hosts. NIH3T3 cells, mouse embryo fibroblasts, were also transformed to anchorage-independent growth in vitro by Int6sh expression. These observations provide direct evidence that the Int6 mutations observed in MMTV-induced tumors and hyperplasia contribute to the malignant transformation of the mammary epithelial cells.

Topics: 3T3 Cells; Agar; Alleles; Animals; Blotting, Northern; Blotting, Western; Breast; Cell Division; Cell Line; Cell Transformation, Neoplastic; Electrophoresis, Polyacrylamide Gel; Epithelial Cells; Eukaryotic Initiation Factor-3; Genes, Dominant; Green Fluorescent Proteins; Humans; Immunohistochemistry; Luminescent Proteins; Mice; Mice, Inbred BALB C; Mutation; Phenotype; Plasmids; Promoter Regions, Genetic; Protein Biosynthesis; Proto-Oncogene Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Time Factors; Transfection; Tumor Cells, Cultured

Rel/NF-kappaB transcription factors control a variety of cellular processes, such as cell growth and apoptosis, that are relevant to oncogenesis, and mutations in genes encoding Rel/NF-kappaB transcription factors have been found in several human lymphoid cell cancers. In this study, we have used a sensitive cell outgrowth assay to demonstrate that wild-type human c-Rel can malignantly transform primary chicken spleen cells, and that transformation by c-Rel is accelerated by co-expression of Bc1-2. Full-length mouse c-Rel can also transform chicken spleen cells. These results are the first demonstration of a lymphoid cell malignant transforming ability for mammalian Rel/NF-kappaB transcription factors, and implicate c-Rel as a molecular target for cancer therapeutics.

Topics: Agar; Animals; Apoptosis; bcl-X Protein; Cell Line, Transformed; Cell Transformation, Neoplastic; Chickens; Colony-Forming Units Assay; Culture Media; Gene Expression Regulation, Neoplastic; Genes, bcl-2; Genes, rel; Humans; Mice; NF-kappa B; Oncogene Proteins v-rel; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-rel; Recombinant Fusion Proteins; Signal Transduction; Species Specificity; Spleen; Transcription, Genetic; Transfection

To search for anti-cancer agents, a screening system for Ras signal inhibitors was developed using a NIH3T3 cell line with an introduced reporter gene which is controlled by the Ras-responsive element (RRE). With this screening system, malolactomycin D was identified as a selective inhibitor of transcription from the RRE. This compound was found to preferentially inhibit the anchorage-independent growth rather than the anchorage-dependent growth of Ras-transformed NIH3T3 cells. The expression of matrix metalloproteinases MMP-1 and MMP-9, which have RRE in their promoters, were reduced by treatment with malolactomycin D at the translational and transcriptional levels. Analysis of the activity of mitogen-activated protein (MAP) kinases, which play important roles in transduction of the Ras signal, showed that malolactomycin D inhibits the activation of p38 MAP kinase and Jun N-terminal-kinase (JNK) but not extracellular signal-regulated kinase 1 or 2 (ERK1 or 2). These findings suggest that by inhibiting the pathway that leads to the activation of p38 MAP kinase and JNK, malolactomycin D suppresses the expression of MMPs. Since MMPs play important roles in metastasis and maintenance of the microenvironment of tumor cells, both of which facilitate tumor growth, the inhibition of MMPs by malolactomycin D is believed to contribute to its ability to inhibit Ras-mediated tumorigenesis.

Topics: 3T3 Cells; Agar; Animals; Anti-Bacterial Agents; Antifungal Agents; Blotting, Northern; Blotting, Western; Cell Division; Cell Transformation, Neoplastic; Dose-Response Relationship, Drug; JNK Mitogen-Activated Protein Kinases; Luciferases; Macrolides; Matrix Metalloproteinase 1; Matrix Metalloproteinase 9; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Neoplasm Metastasis; p38 Mitogen-Activated Protein Kinases; Promoter Regions, Genetic; ras Proteins; Time Factors; Transcription, Genetic; Transfection

Research into molecular and genetic mechanisms underlying prostate carcinogenesis would be greatly advanced by in vitro models of prostate tumors representing primary tumors. We have successfully established an immortalized human prostate epithelial (HPE) cell culture derived from a primary tumor with telomerase. The actively proliferating early passaged RC-58T cells were transduced through infection with a retrovirus vector expressing the human telomerase catalytic subunit (hTERT). A high level of telomerase was detected in RC-58T/hTERT cells but not RC-58T cells. RC-58T/hTERT cells are currently growing well at passage 50, whereas RC-58T cells senesced at passage 7. RC-58T/hTERT cells exhibit transformed morphology. More importantly, these immortalized cells showed anchorage-independent growth as they formed colonies in soft agar and grew above the agar layer. Expression of androgen-regulated prostate specific gene NKX3.1 and epithelial specific cytokeratin 8 (CK8) but not prostate specific antigen (PSA) and androgen receptor was detected in RC-58T/hTERT cells. Prostate stem cell antigen (PSCA) and p16 were also expressed in this cell line. RC-58T/hTERT cells showed growth inhibition when exposed to retinoic acid and transforming growth factor (TGF)-beta1 known potent inhibitors of prostate epithelial cell growth. A number of chromosome alterations were observed including the loss of chromosomes Y, 3p, 10p, 17p, 18q and the gain of chromosomes 16 and 20. These results demonstrate that this primary tumor-derived HPE cell line retained its transformed phenotypes and should allow studies to elucidate molecular and genetic alterations involved in prostate cancer. This is the first documented case of an established human prostate cancer cell line from a primary tumor of a prostate cancer patient with telomerase.

Topics: Agar; Cell Culture Techniques; Cell Division; Cell Line, Transformed; Cell Size; Cell Transformation, Neoplastic; Chromosome Aberrations; DNA-Binding Proteins; Epithelial Cells; Humans; Karyotyping; Male; Models, Biological; Prostatic Neoplasms; Retroviridae; Reverse Transcriptase Polymerase Chain Reaction; RNA, Neoplasm; Telomerase; Transduction, Genetic; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tretinoin; Tumor Cells, Cultured

During attempts to transform a normal human fibroblast strain (GM730) by X-irradiation, we obtained a partially transformed cell strain (GM730pt) which demonstrates several aspects of the transformed phenotype including morphological changes, increased saturation density, growth in soft agar, and focus formation in long-term cultures. When GM730pt cells were transfected with the feline c-myc gene, morphology of the cells changed dramatically following seven days of expression. Transfection of other plasmid DNAs or oncogenes such as pUC8, pSV2neo, src, sis, and H-ras had little or no effects on the phenotype of GM730pt cells. On the other hand, a gel purified, small fragment of c-myc DNA had a complete cell alteration activity. Furthermore, Bal 31 deletion and M13 sequencing experiments showed that the alteration seen in GM730pt cells is delimited to a 24 nucleotide stretch (active myc element) from the second intron of the feline c-myc gene that contains a T-rich sequence.

Topics: Agar; Animals; Base Sequence; Blotting, Southern; Cats; Cell Count; Cell Division; Cell Line, Transformed; Cell Size; Cell Transformation, Neoplastic; Cells, Cultured; Fibroblasts; Genes, myc; Humans; Infant, Newborn; Introns; Male; Middle Aged; Oncogenes; Plasmids; Poly T; Sequence Deletion; Time Factors; Transfection; Tumor Stem Cell Assay

Strategies that antagonize growth factor signaling are attractive candidates for the biological therapy of brain tumors. HGF/NK2 is a secreted truncated splicing variant and potential antagonist of scatter factor/hepatocyte growth factor (SF/HGF), a multifunctional cytokine involved in the malignant progression of solid tumors including glioblastoma. U87 human malignant glioma cells that express an autocrine SF/HGF stimulatory loop were transfected with the human HGF/NK2 cDNA and clonal cell lines that secrete high levels of HGF/NK2 protein (U87-NK2) were isolated. The effects of HGF/NK2 gene transfer on the U87 malignant phenotype were examined. HGF/NK2 gene transfer had no effect on 2-dimensional anchorage-dependent cell growth. In contrast, U87-NK2 cell lines were approximately 20-fold less clonogenic in soft agar and approximately 4-fold less migratory than control-transfected cell lines. Intracranial tumor xenografts derived from U87-NK2 cells grew much slower than controls. U87-NK2 tumors were approximately 50-fold smaller than controls at 21 days post-implantation and HGF/NK2 gene transfer resulted in a trend toward diminished tumorigenicity. This report shows that the predominant effect of transgenic HGF/NK2 overexpression by glioma cells that are autocrine for SF/HGF stimulation is to inhibit their malignant phenotype.

Topics: Agar; Alternative Splicing; Animals; Autocrine Communication; Brain Neoplasms; Cell Adhesion; Cell Division; Cell Movement; Cell Transformation, Neoplastic; Gene Transfer Techniques; Glioma; Hepatocyte Growth Factor; Humans; Mice; Mice, SCID; Molecular Weight; Neoplasm Transplantation; Phenotype; Transfection; Transplantation, Heterologous; Tumor Cells, Cultured

Topics: 3T3 Cells; Agar; Animals; Cell Adhesion; Cell Transformation, Neoplastic; Culture Media; Guanine Nucleotide Exchange Factors; Mice; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-raf; rac1 GTP-Binding Protein; rho GTP-Binding Proteins; Rho Guanine Nucleotide Exchange Factors

The Tel gene is a major target of translocations in leukemia and loss of heterozygosity is regularly observed for the non-translocated allele, thus supporting the notion that Tel is a tumor suppressor. Most tumor suppressors influence cellular proliferation, differentiation and cell death and thereby prevent oncogenic transformation and genetic instability. We found that overexpression of Tel retards proliferation of many cell types, primary cells and immortalized cells, by inducing a G1 arrest. Tel's block of cellular proliferation is rescued by high seeding densities. Furthermore, Tel suppressed Ras-mediated colony growth in soft agar and tumor formation in nude mice. The Pointed and DNA binding (DB) domains of Tel were required for all Tel-induced phenotypes.

Topics: 3T3 Cells; Agar; Animals; Blotting, Western; Cell Count; Cell Division; Cell Line, Transformed; Cell Transformation, Neoplastic; Cells, Cultured; DNA-Binding Proteins; ETS Translocation Variant 6 Protein; Fibroblasts; Flow Cytometry; Fluorescent Antibody Technique; G1 Phase; Genes, ras; Mice; Mice, Inbred C57BL; Mice, Nude; Oncogene Protein p21(ras); Protein Structure, Tertiary; Proto-Oncogene Proteins c-ets; Repressor Proteins; Transcription Factors; Tumor Stem Cell Assay

We have previously found that epidermal growth factor (EGF) mediates growth through the Jun N-terminal kinase/stress-activated kinase (JNK/SAPK) pathway in A549 human lung carcinoma cells. As observed here, EGF treatment also greatly enhances the tumorigenicity of A549 cells, suggesting an important role for JNK in cancer cell growth (F. Bost, R. McKay, N. Dean, and D. Mercola, J. Biol. Chem. 272:33422-33429, 1997). Several isoforms families of JNK, JNK1, JNK2, and JNK3, have been isolated; they arise from alternative splicing of three different genes and have distinct substrate binding properties. Here we have used specific phosphorothioate oligonucleotides targeted against the two major isoforms, JNK1 and JNK2, to discriminate their roles in EGF-induced transformation. Multiple antisense sequences have been screened, and two high-affinity and specific candidates have been identified. Antisense JNK1 eliminated steady-state mRNA and JNK1 protein expression with a 50% effective concentration (EC50) of <0.1 microM but did not alter JNK2 mRNA or protein levels. Conversely, antisense JNK2 specifically eliminated JNK2 steady-state mRNA and protein expression with an EC50 of 0.1 microM. Antisense JNK1 and antisense JNK2 inhibited by 40 and 70%, respectively, EGF-induced total JNK activity, whereas sense and scrambled-sequence control oligonucleotides had no effect. The elimination of mRNA, protein, and JNK activities lasted 48 and 72 h following a single Lipofectin treatment with antisense JNK1 and JNK2, respectively, indicating sufficient duration for examining the impact of specific elimination on the phenotype. Direct proliferation assays demonstrated that antisense JNK2 inhibited EGF-induced doubling of growth as well as the combination of active antisense oligonucleotides did. EGF treatment also induced colony formation in soft agar. This effect was completely inhibited by antisense JNK2 and combined-antisense treatment but not altered by antisense JNK1 alone. These results show that EGF doubles the proliferation (growth in soft agar as well as tumorigenicity in athymic mice) of A549 lung carcinoma cells and that the JNK2 isoform but not JNK1 is utilized for mediating the effects of EGF. This study represents the first demonstration of a cellular phenotype regulated by a JNK isoform family, JNK2.

Topics: Agar; Animals; Calcium-Calmodulin-Dependent Protein Kinases; Cell Division; Cell Transformation, Neoplastic; Epidermal Growth Factor; Female; Gene Expression; Humans; Isoenzymes; JNK Mitogen-Activated Protein Kinases; Mice; Mice, Nude; Mitogen-Activated Protein Kinase 9; Mitogen-Activated Protein Kinases; Oligonucleotides; Oligonucleotides, Antisense; Protein Kinases; RNA, Antisense; RNA, Messenger; Time Factors; Tumor Cells, Cultured; Ultraviolet Rays

The chemokine receptor CXCR2 is the closest homologue to Kaposi's sarcoma herpesvirus-G protein-coupled receptor (KSHV-GPCR), which is known to be constitutively activated and able to cause oncogenic transformation. Among G protein-coupled receptors, a DRY sequence in the second intracellular loop is highly conserved. However, the KSHV-GPCR shows a VRY sequence instead. In this study, we exchanged Asp138 of the DRY sequence in the CXCR2 with a Val (D138V), the corresponding amino acid in KSHV-GPCR, or with a Gln (D138Q), and investigated the functional consequences of these mutations. In focus formation and soft agar growth assays in NIH 3T3 cells, the D138V mutant exhibited transforming potential similar to the KSHV-GPCR. Surprisingly, the CXCR2 wild type itself showed transforming activity, although not as potently, due to continuous autocrine stimulation, whereas the D138Q mutant formed no foci. In agreement with these results were high levels of inositol phosphate accumulation in the D138V mutant and the KSHV-GPCR, indicating constitutive activity. These data emphasize the importance of the DRY sequence for G protein-coupled signaling of the CXCR2. Either constitutive activation or persistent autocrine stimulation of the CXCR2 causes transformation similar to KSHV-GPCR-transfected cells, probably activating the same signal transduction cascade that can abrogate normal growth control mechanisms.

Topics: 3T3 Cells; Actins; Agar; Amino Acid Sequence; Animals; Calcium; Cell Division; Cell Transformation, Neoplastic; Chemokines, CXC; Contact Inhibition; Herpesvirus 8, Human; Humans; Inositol Phosphates; Mice; Molecular Sequence Data; Point Mutation; Rats; Receptors, Chemokine; Sarcoma, Kaposi; Signal Transduction; Tumor Cells, Cultured

Although there is indirect genetic evidence that MEN1, the gene for multiple endocrine neoplasia type 1, is a tumor suppressor gene, little is known about the MEN1-encoded protein, menin. Menin was stably overexpressed in a well-characterized murine tumor cell line, (valine-12)-RAS-transformed NIH3T3 cells. Menin overexpression reverted the morphology of the RAS-transformed NIH3T3 cells towards the more flattened and more spread, fibroblastic shape of wild type NIH3T3 cells. The proliferation rate of the RAS-transformed cells in 0.5% calf serum was also slower with menin overexpression. Menin overexpression reduced the RAS-induced clonogenicity in soft agar. Menin also reduced tumor growth after injection of cells in nude mice. In conclusion, stable overexpression of MEN1 suppressed partially the RAS-mediated tumor phenotype in vitro and in vivo. Overexpressed menin protein had biological effects, directly supporting MEN1 gene function as a tumor suppressor.

Topics: 3T3 Cells; Agar; Amino Acid Sequence; Animals; Cell Division; Cell Nucleus; Cell Transformation, Neoplastic; Cells, Cultured; Culture Media; Fibroblasts; Gene Expression Regulation; Genes, Tumor Suppressor; Humans; Mice; Mice, Nude; Molecular Sequence Data; Multiple Endocrine Neoplasia Type 1; Neoplasm Proteins; Proto-Oncogene Proteins; ras Proteins; Transfection

The MLEC10 is an epithelial cell line derived from an untreated, normal C3H/HeN mouse liver. We previously demonstrated that tumorigenic variants from this cell line produced moderately differentiated hepatocellular carcinomas in nude mice. However, it has remained unclear whether the parental MLEC10 cells represent immortalized hepatocytes or so-called oval cells, both of which may serve as precursors for hepatocellular neoplasms. In this study, we performed 3-dimensional, spheroid culture of the MLEC100 cells in order to facilitate histological assessment of their lineage. Spheroidal aggregates were formalin-fixed and embedded in paraffin for routine light-microscopic observation of hematoxylin and eosin-stained sections. Histopathologically, the MLEC10 cells were indistinguishable from immature hepatocytes and distinct from oval cells. At the electron-microscopic level, their hepatocytic nature was evidenced by bile canaliculus structures and glycogen storage. Intriguingly, the spheroids contained fragmentary material reminiscent of Councilman bodies, implying apoptosis of the hepatocytes. Although the cells significantly proliferated during the first three days of culture, apoptotic death then resulted in a 75 % decrease in viable cell number. Thereafter, both apoptosis and cell division appeared silent, the numbers being unchanged. Expression of the p53 tumor suppressor gene became gradually elevated, correlating positively with growth arrest, but negatively with apoptosis, suggesting that the cell death occurred independently of p53. Our results indicate that at least some liver epithelial cell lines derived from untreated murine livers exhibit a hepatocytic morphology in spheroid culture. Also, the present culture system provides a useful tool for investigating biological phenomena, e.g. apoptosis, specifically involving liver cells, under 3-dimensional conditions.

Topics: Agar; Animals; Apoptosis; Cell Culture Techniques; Cell Division; Cell Line; Cell Survival; Cell Transformation, Neoplastic; Epithelial Cells; Gene Expression Regulation; Genes, myc; Genes, p53; Genes, ras; Humans; Kinetics; Liver; Male; Mice; Mice, Inbred C3H; Time Factors; Transfection

The contribution of the ETS2 transcription factor to the transformed state in prostate cancer cells has been assessed. Northern blot analysis easily detects ETS2 in DU145 and PC3, high grade human prostate cell lines, but ETS2 is not present in lower grade LNCaP cells. Stable transfection of PC3 and DU145 prostate cell lines with an antisense ETS2 vector or with a dominant negative ETS2 mutant significantly reduced the ability of DU145 and PC3 cells to form large colonies in soft agar. Thus, the presence of ETS2 is positively correlated with a more transformed phenotype and blockage of ETS2 function can reduce transformed properties of prostate cancer cells.

Topics: Agar; Androgens; Blotting, Northern; Cell Division; Cell Transformation, Neoplastic; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Genes, Dominant; Humans; Male; Phenotype; Precipitin Tests; Prostatic Neoplasms; Proto-Oncogene Protein c-ets-2; Proto-Oncogene Proteins; Repressor Proteins; RNA, Antisense; Sequence Deletion; Trans-Activators; Transcription Factors; Transfection; Tumor Cells, Cultured

Anchorage-independent growth is characteristic of neoplastic cells, but the signal transduction pathways that mediate this phenotype are poorly understood. Several important cell cycle events are dependent on cell-substratum adhesion in non-transformed cells, including activation of G1 cyclin-dependent kinases and expression of cyclin A; the adhesion requirement of these events is abrogated in Ras-transformed cells. The ER-1-2 mutant rat fibroblast cell line is: 1) resistant to Ras-mediated, anchorage-independent growth; 2) defective in Ras-mediated, adhesion-independent expression of cyclin A, but not adhesion-independent activation of cyclin-dependent kinases; and 3) rescued for Ras-induced, anchorage-independent growth by ectopic expression of cyclin A. We report here that extracellular ATP induces adhesion-independent expression of cyclin A and rescues growth in soft agar by ER-1-2 cells that express Ras. ADP, AMP and the non-hydrolyzable analog adenosine 5'-(beta, gamma-iminodiphosphate) are also effective, but adenosine is not. Adenine nucleotide-induced growth in soft agar is inhibited by reactive blue 2, an antagonist of some P2 purinoceptors. ATP does not induce adhesion-independent expression of cyclin A in ER-1-2 or control rat fibroblasts that do not express Ras, indicating a requirement for additional Ras-regulated signals for expression of this gene; one such signal may lead to phosphorylation of the retinoblastoma protein, pRB, and related proteins. These results suggest that extracellular ATP could play a role in the multistage carcinogenic process in vivo.

Topics: Adenine Nucleotides; Adenosine; Adenosine Triphosphate; Agar; Animals; Cell Adhesion; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cyclins; Genes, ras; Mutagenesis; Protein Synthesis Inhibitors; Purinergic P2 Receptor Antagonists; ras Proteins; Rats; Triazines

Using a novel strategy that enables the isolation of previously unknown genes encoding selectable recessive phenotypes, we identified a gene (tsg101) whose homozygous functional disruption produces cell transformation. Antisense RNA from a transactivated promoter introduced randomly into transcribed genes throughout the genome of mouse 3T3 fibroblasts was used to knock out alleles of chromosomal genes adjacent to promoter inserts, generating clones that grew in 0.5% agar and formed metastatic tumors in nude mice. Removal of the transactivator restored normal growth. The protein encoded by tsg101 cDNA encodes a coiled-coil domain that interacts with stathmin, a cytosolic phosphoprotein implicated previously in tumorigenesis. Overexpression of tsg101 antisense transcripts in naive 3T3 cells resulted in cell transformation and increased stathmin-specific mRNA.

Topics: 3T3 Cells; Agar; Alleles; Amino Acid Sequence; Animals; Base Sequence; Cell Differentiation; Cell Division; Cell Transformation, Neoplastic; Chromosome Mapping; Cloning, Molecular; Cytosol; DNA-Binding Proteins; DNA, Complementary; Endosomal Sorting Complexes Required for Transport; Gene Expression Regulation, Neoplastic; Genes, Recessive; Genes, Tumor Suppressor; Homozygote; Mammals; Mice; Mice, Knockout; Mice, Nude; Microtubule Proteins; Molecular Sequence Data; Neoplasms, Experimental; Phenotype; Phosphoproteins; RNA, Antisense; RNA, Messenger; Sequence Analysis, DNA; Stathmin; Transcription Factors; Transformation, Genetic

We previously identified Fgf-8 as a frequently activated gene in tumors from mouse mammary tumor virus-infected Wnt-1 transgenic mice, suggesting that Fgf-8 is a proto-oncogene. We further determined that multiple, secreted protein isoforms that differ at their mature amino termini are encoded by alternatively spliced mRNAs transcribed from the gene. We now present evidence that there are differences in the potency of NIH3T3 cell transformation displayed by three of the FGF (fibroblast growth factor)-8 isoforms. We find that stable transfection of a cDNA for the FGF-8b isoform leads to marked morphological transformation of NIH3T3 cells and rapid tumorigenicity of the transfected cells in nude mice. In contrast, transfection of a cDNA for the FGF-8a or FGF-8c isoform results in moderate morphological changes in the NIH3T3 cells, and the transfected cells are weakly tumorigenic in nude mice. All three transfections result in cells that express comparable amounts of Fgf-8 mRNA and that produce the FGF-8 protein isoforms. The morphological changes observed in NIH3T3 cells can be reproduced by the addition of recombinant FGF-8 protein isoforms to the culture medium. Therefore, these results indicate that there are differences in the potency of transformation of NIH3T3 cells by FGF-8 protein isoforms and suggest that these FGF-8 isoforms may have different in vivo functions.

Topics: 3T3 Cells; Agar; Animals; Cell Size; Cell Transformation, Neoplastic; DNA, Complementary; Fibroblast Growth Factor 8; Fibroblast Growth Factors; Gene Expression; Growth Substances; Isomerism; Mice; Neoplasm Proteins; Proto-Oncogenes; Recombinant Proteins; RNA, Messenger; Transfection

Considerable progress has been made in elucidating the components of the Ras signalling pathway, from both biochemical and genetic investigations. However little is known about the nuclear targets of the pathway, and in particular those that mediate the long-term changes in gene expression resulting from Ras transformation. Ets family members may be involved in these processes since Ras stimulates transcription through ets-DNA binding sites. We show that a mutated Ets protein, delta PU.1, inhibits Ras activation of transcription. Stable expression of delta PU.1 in Ras transformed NIH3T3 fibroblasts reverts the transformed phenotype by many characteristics, including morphology, anchorage independent growth, saturation density, growth in low serum, tumour formation in nude mice and to some extent sensitivity to apoptotic cell death. Similar trans-dominant mutants of c-Ets-1 and c-Ets-2, the most divergent members of the Ets-family to PU.1, also revert Ras transformed cells, as indicated by morphology, anchorage-independent growth, saturation density and doubling time in low serum. Reversion may result from a shared property of the mutants, such as binding to ets motifs in promoters. These results provide evidence for an important role for Ets proteins in Ras transformation.

Topics: 3T3 Cells; Agar; Animals; Blood; Cathepsin L; Cathepsins; Cell Transformation, Neoplastic; Cysteine Endopeptidases; Endopeptidases; Genes, Dominant; Genes, p53; Genes, ras; Genes, Retinoblastoma; Mice; Mutation; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-ets; RNA, Messenger; Sequence Deletion; Signal Transduction; Transcription Factors; Transcriptional Activation

Morphofunctional peculiarities of tumor cells from 15 endometrial adenocarcinomas and 2 ovarian tumors have been investigated at the ultrastructural level. These cells could develop two types of colonies in soft agar: those with histotypical differentiation (numerous microvilli, well developed tight junctions, desmosomes, secretory granules), and those without it (absence of epithelial features, ability of tumor cells to produce filamentous extracellular matrix and striated collagen fibrils which are characteristic of fibroblastic cells). The addition of progesterone and tamoxifen to cell cultures resulted in rising the level of cell differentiation in the colonies. The fact that endometrial and ovarian cancer cells can express the properties specific of connective tissue cells may suggest a multipotention of the Mullerian epithelium derivatives to shed light on the histogenesis of the mixed Mullerian tumors of uterus.

Topics: Adenocarcinoma; Agar; Cell Transformation, Neoplastic; Culture Media; Cystadenocarcinoma; Endometrial Neoplasms; Female; Granulosa Cell Tumor; Humans; Methotrexate; Microscopy, Electron; Morphogenesis; Ovarian Neoplasms; Progesterone; Tamoxifen; Tumor Cells, Cultured

Expression of the early regions of several primate polyomaviruses (SV40, BKV, JCV, and LPV) in hamster cells induces transformation, manifested by the ability to grow in soft agar. Hamster cells transformed by SV40 contain complexes between the SV40 T antigen and the cellular tumor suppressor protein p53. We detected analogous complexes between p53 and the BKV T antigen in hamster cells transformed by the BKV early region, where the half life of p53 increased 16-fold. However, neither a LPV-transformed hamster fibroblast cell line [LPV-HE (F); K. K. Takemoto and T. Kanda, 1984, J. Virol. 50, 100-105] nor BHK-21 cells transformed by the LPV early region contained detectable complexes between the LPV T antigen and p53, nor was the stability of p53 in LPV transformed BHK-21 cells altered. Association between hamster p53 and the LPV T antigen expressed as glutathione S-transferase fusion protein could not be detected in vitro. These data indicate that alteration of the amount or stability of p53 is not required for transformation of hamster cells by LPV. However, as viruses such as SV40 and BKV whose T antigens bind p53 are oncogenic in hamsters, whereas LPV is not, the alteration of p53 amount or stability may be required for tumorigenesis.

Topics: Agar; Animals; Antigens, Viral, Tumor; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cricetinae; Culture Media; Glutathione Transferase; Lymphoid Tissue; Organ Specificity; Polyomavirus; Transcription, Genetic; Tumor Suppressor Protein p53

The ultrastructure of cells from seven human lung cancers and from the colonies formed by these cells in soft agar was investigated. Tumor cells developed to display the morphofunctional potentials of the initial tumors. Cultured cells of squamous-cell carcinomas contained numerous tonofilaments, those of adenocarcinomas developed microvilli on their apical surfaces and intracellular lumens. On the other hand, cells of squamous-cell carcinomas showed features specific of adenoma epithelium, i.e. well developed microvilli and intracellular lumens. Besides, cells of adenocarcinoma often contained large quantities of tonofilaments considered to be characteristic of epidermoid epithelium. The results obtained suggest a possibility of metaplastic transformation of the lung epithelium.

Topics: Adenocarcinoma; Adenocarcinoma, Bronchiolo-Alveolar; Agar; Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; Culture Media; Humans; Lung Neoplasms; Methotrexate; Microscopy, Electron; Morphogenesis; Prospidium; Tumor Cells, Cultured; Vincristine

Upon neoplastic transformation, cells acquire the ability to grow in soft agar. We investigated how this occurs by cell cycle analysis of a rat cell line NRK-49F and its transformation-deficient mutants. Rapidly growing NRK and mutants arrest in G1 when deprived of anchorage by suspending in methylcellulose. Addition of epidermal growth factor (EGF) together with transforming growth factor-beta (TGF-beta), which is highly oncogenic to NRK, induces the rapid progression of G1-arrested NRK cells into S phase. The time course and the extent of synchronization are very similar to the cell cycle progression in the presence of anchorage. EGF alone, which is highly mitogenic but only slightly oncogenic, fails to induce such progression. Both mutants remain arrested in G1. These data indicate that oncogenic signals confer on NRK the ability to enter S phase in the absence of anchorage and that this is the principal mechanism for its ability to grow in soft agar.

Topics: Agar; Animals; Cell Adhesion; Cell Division; Cell Line; Cell Transformation, Neoplastic; Gene Expression; Genes, fos; Genes, myc; Mutation; Oncogenes; S Phase; Signal Transduction

Automated image analysis was used to measure the colony diameter and estimate the cell number in colonies under conditions for anchorage-independent growth (AIG). The calculated values for the number of AIG cells in a colony of a diameter greater than or equal to 60 microns were decidedly different from the actual values obtained by either automated or manual cell counting. Like animal cells that exhibit AIG, these colonies demonstrating anchorage independence can be enumerated reproducibly. However, the assumption is incorrect that the number of cells (N) in a colony expressing AIG varies directly with a change in the diameter of a colony (d). Whether one counts the number of cells manually in a colony of a definite diameter or arbitrarily uses a diameter function of greater than or equal to 60 microns as a linear distance that indicates the presence of 50 or more cells in a colony, each procedure has its own built-in bias. In procedures that require quantitative data, the most reliable procedure is to standardize the values for diameter of colonies with cell numbers in colonies.

Topics: Agar; Cell Division; Cell Transformation, Neoplastic; Humans; Image Processing, Computer-Assisted; Mathematics; Microscopy, Fluorescence; Reproducibility of Results; Spectrophotometry, Ultraviolet

The proto-oncogene c-myc and the oncogene SV40T, both of which have been implicated in the process of cellular immortalization in vitro, have been introduced via amphotropic retroviral expression vectors into the human mammary epithelial cell (HMEC) line 184A1N4 (A1N4). Two stable cell lines were established by growth in selective medium and were found to overexpress either c-myc (A1N4-myc) or SV40T antigen (A1N4-T). Neither the A1N4, A1N4-myc, or A1N4-T cells will grow in soft agar or form tumors in nude mice. However, A1N4-T or A1N4-myc cells, but not the parental A1N4 cells, form colonies in soft agar in response to either epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha), or basic fibroblast growth factor (bFGF). Like EGF and TGF alpha, bFGF is moderately mitogenic for the anchorage-dependent growth (ADG) of all three cell lines. Further, co-cultivation of A1N4-T or A1N4-myc cells with primary diploid mammary fibroblasts can also induce the anchorage-independent growth (AIG) and stimulate the ADG of A1N4-T or A1N4-myc. In addition, conditioned medium obtained from these mammary fibroblasts also stimulated the AIG of the A1N4-T and A1N4-myc cells and was found to contain immunoreactive TGF alpha and bioactive FGF. The mammary fibroblasts express specific mRNA transcripts for bFGF and acidic FGF (aFGF). These results suggest that growth factors such as TFG alpha or FGF, which may be derived from the adjacent mammary stroma, might influence in a paracrine manner the phenotypic characteristics of a population of human mammary epithelial cells toward transformation.

Topics: Agar; Antigens, Viral, Tumor; Breast; Cell Division; Cell Line, Transformed; Cell Membrane; Cell Transformation, Neoplastic; Epidermal Growth Factor; Epithelium; ErbB Receptors; Fibroblast Growth Factor 2; Fibroblast Growth Factors; Fibroblasts; Humans; Proto-Oncogene Mas; Proto-Oncogene Proteins c-myc; Receptors, Cell Surface; Receptors, Fibroblast Growth Factor; Simian virus 40; Transforming Growth Factor alpha

Topics: Agar; Animals; Cell Adhesion; Cell Transformation, Neoplastic; Dose-Response Relationship, Drug; Epithelium; Mice; Neoplastic Stem Cells; Tumor Cells, Cultured

The eph gene encodes a putative receptor tyrosine kinase for an as yet unknown ligand. Some human cancer cells have been found to overexpress eph mRNAs without gene amplification. We show here that NIH3T3 cells acquire tumorigenic ability in nude mice and make colonies in soft agar with a viral LTR (Long Terminal Repeat)-driven artificial expression of the eph gene to a high level. This result supports the alleged contribution of overexpressed receptor tyrosine kinases to cell transformation.

Topics: Agar; Amino Acid Sequence; Animals; Blotting, Southern; Cell Adhesion; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Genes; Mice; Mice, Nude; Molecular Sequence Data; Protein-Tyrosine Kinases; Repetitive Sequences, Nucleic Acid; Transfection

Seven temperature-sensitive (ts) mutants of Abelson murine leukaemia virus (A-MuLV) were isolated on the basis of the temperature dependence of their soft agar colony-forming ability. These seven ts mutants exhibited similar characteristics and were not ts for morphological transformation and autophosphorylation of P120gag-abl protein. The dissociation of the properties of morphology, soft agar colony formation and tyrosine kinase activity might suggest that the v-abl product has more than one primary intracellular target.

Topics: Abelson murine leukemia virus; Agar; Animals; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cells, Cultured; Leukemia Virus, Murine; Mice; Mutation; Phosphorylation; Protein-Tyrosine Kinases; Temperature

We have developed a hybrid methylcellulose/agar suspension culture system which permits long-term colony formation of transformed mesenchymal cells. In contrast to traditional agar suspensions, our system allows for recovery of cells and direct biochemical analysis of anchorage-independent growth. The ability to readily radiolabel cellular macromolecules in these preparative cultures permits a quantitative and objective analysis of colony formation by incorporation of [3H]thymidine into newly synthesized DNA.

Topics: Agar; Cell Count; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Culture Media; DNA Replication; Gentian Violet; Methylcellulose; Phenotype; Suspensions; Thymidine; Tritium

Transient (about 2 hr) acidification to approx. pH 5.0 of agar-gelled overlayers containing untransformed NRK-49F or KiMSV-transformed NRK-49F cells in the presence of fetal calf serum or crude 49F-cell conditioned medium, as sources of latent TGF-beta, elicited EGF-dependent colony formation of 49F cells and inhibited spontaneous growth of transformed cells. Pure, active TGF-beta (porcine, type I) had the same effects on these respective cell types, suggesting that the above results were due to activation of latent TGF-beta in the transiently acidic cellular environment. Similar acidifications in the absence of a source of latent TGF-beta enhanced the positive growth response of 49F and AKR-2B cells to EGF and active TGF-beta and also the negative growth response of KiMSV-transformed 49F cells to active TGF-beta. These results are compatible with the idea that acidic cellular environments, particularly in tumor tissues, are conducive to activation of latent TGF-beta, perhaps in conjunction with other activating mechanisms, and to an enhanced response to some growth factors. However, the heterogeneity of cell populations within tumoral masses presents an obstacle to a clear understanding of the consequences of such activation.

Topics: Agar; Animals; Blood; Cell Division; Cell Line; Cell Transformation, Neoplastic; Culture Media; Epidermal Growth Factor; Hydrogen-Ion Concentration; Lactates; Lactic Acid; Mice; Transforming Growth Factors

HL-60 cells differentiate primarily to eosinophils instead of neutrophils when cultured with butyric acid if they have previously been cultured under alkaline conditions (pH 7.6). To determine the nature of the process by which this occurs, a group of single-cell derived clones was produced from HL-60 cells after prolonged passage under alkaline conditions. When these clones were induced to mature with butyric acid, each clone demonstrated a characteristic proportion of mature eosinophils and neutrophils. This property was stable for multiple passages. Subclones derived from these clones also demonstrated the same probability of differentiating to an eosinophil as their parent clones. Reversion toward neutrophilic differentiation gradually occurred after several months of culture under conditions of reduced pH. The most highly directed clones demonstrated 90-95% eosinophilic differentiation and continued to differentiate primarily to eosinophils after seven months of culture at the reduced pH. Thus, in HL-60 cells, the tendency to differentiate to an eosinophil is a long-lived, heritable, continuously variable phenotype that is inducible in cells by culture under alkaline conditions. This tendency persists for prolonged periods after the alkaline conditions are removed, but may gradually revert toward neutrophilic differentiation with time.

Topics: Agar; Butyrates; Butyric Acid; Cell Differentiation; Cell Line; Cell Transformation, Neoplastic; Culture Media; Eosinophils; Humans; Hydrogen-Ion Concentration; Leukemia, Promyelocytic, Acute; Probability; Tumor Cells, Cultured; Tumor Stem Cell Assay

Seeding 2 X 10(4) cells from a clone of BALB/c 3T3 cells in agar led to the formation of about 100 small colonies (approximate diameter, 0.2 mm) and two large colonies (1-mm diameter). Seven of the former and both of the latter were isolated, and the morphology and growth properties of their cells were observed in repeated weekly passages. The seven subclones derived from the smaller agar colonies spread out on the dish and multiplied more slowly than the parental clone on plastic, but six of them produced more colonies in agar than the parental clone. The two subclones derived from the larger agar colonies had a fully transformed morphology, multiplied much faster than the parental clone on plastic, and produced a high percentage of large colonies in agar. Each of the subclones could be distinguished morphologically from the others and from the parental clone, and most of them could be distinguished on the basis of their colony-forming efficiency in agar. Most of the secondary subclones derived from an early passage on plastic of one of the two large agar clones, died out on the second passage after their isolation. Secondary subclones derived from the same subclone five weeks later had a wide range of fluctuating growth rates, but did not die out on passage. The two rapidly growing subclones derived from large agar colonies initiated fast-growing tumors in nude mice. None of the other subclones produced any visible growth in nude mice over a 3-month period. Large agar colonies of fast growing, morphologically transformed cells appeared once during further passage of the parental clone and two of the subclones. The results reveal a surprising degree of heritable diversity in morphology and growth characteristics of the progeny from a clonal line of nontransformed cells. They also indicate that, in this cell line, only those cells that have a high efficiency (greater than 20%) of large colony formation in agar have the capacity to form tumors in nude mice.

Topics: Agar; Animals; Cell Transformation, Neoplastic; Cells, Cultured; Clone Cells; Mice; Mice, Inbred BALB C; Mice, Nude

We developed a method to determine the amount of work performed by cells through cell division in 1.0% agar cultures. There was no correlation between the cloning efficiencies of 1.0 and 0.3% agar cultures. Growth in 1.0% agar cultures correlated well with such malignant properties as tumorigenicity and the invasive and metastatic potentials. Our method revealed that metastatic MC and F cell lines possess different means of taking advantage of energy to proliferate against an environmental pressure from those possessed by nontumorigenic (ME and T-C3H) cell strain/line or nonmetastatic but tumorigenic (L,MR, and magc1) cell lines.

Topics: Agar; Animals; Cell Line; Cell Transformation, Neoplastic; Clone Cells; Culture Media; Elasticity; Mice; Mice, Inbred A; Mice, Inbred C3H; Models, Biological; Muscles; Neoplasm Metastasis; Organ Culture Techniques; Pressure; Tumor Cells, Cultured

The establishment of a spontaneously transformed tumorigenic human fibrosarcoma line derived from the ovary of a breast cancer patient (NO-15) is described. This tumor cell line has been characterized by tumor growth in nude mice. The cells form colonies without the usually recommended agar technique and are sensitive to cis-dichlorodiammineplatinum(II) (cis-DDP), daunorubicin (DR) and bleomycin (BLM), three well-known chemotherapeutic agents. The cells were incubated in the presence of drugs using three different methods. These agents proved to be noncytotoxic to monolayers treated for three hours. Conversely dose-dependent cytotoxicity was seen in suspension cultures treated for three hours. Optimal correlation between the three methods for the evaluation of cytotoxicity was seen in monolayers exposed to these agents for 24 hours. The results of three different methods to evaluate drug efficacy (number of colonies, growth rate and 3H-thymidine incorporation) were in good correlation. The incorporation of the radiolabeled tracer into cellular DNA is advantageous for its rapidity and quantitative reproducibility.

Topics: Adult; Agar; Animals; Antineoplastic Agents; Breast Neoplasms; Cell Line, Transformed; Cell Transformation, Neoplastic; Colony-Forming Units Assay; Culture Media; Female; Humans; Mice; Mice, Nude; Neoplasm Transplantation; Ovary; Tumor Cells, Cultured; Tumor Stem Cell Assay

Topics: Agar; Animals; Cell Transformation, Neoplastic; Cells, Cultured; Colony-Forming Units Assay; Culture Media; Humans; Mice; Neoplasm Transplantation; Tumor Stem Cell Assay; Urogenital Neoplasms

Topics: Agar; Animals; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Clone Cells; Lung Neoplasms; Mammary Neoplasms, Experimental; Mice; Neoplasm Metastasis; Neoplastic Stem Cells; Rats; Tumor Stem Cell Assay

Acrylonitrile (AN) and acrylamide (AM) are carcinogenic in a number of rodent organs and AN is a suspected human carcinogen. We sought to determine whether AN and/or AM could produce morphological transformation in vitro in C3H/10T1/2 and NIH/3T3 mouse fibroblast cells. Both AN and AM induced a dose-dependent cytotoxic effect in C3H/10T1/2 and NIH/3T3 cells and readily transformed both cell lines. Our conclusions are based on the appearance of cells exhibiting a transformed phenotype and growth in soft agar. AN and AM transformed NIH/3T3 cells to a greater extent than C3H/10T1/2 cells. This is the first reported transformation of cells in vitro by AM.

Topics: Acrylamide; Acrylamides; Acrylonitrile; Agar; Animals; Cell Line; Cell Survival; Cell Transformation, Neoplastic; DNA; Dose-Response Relationship, Drug; Mice; Nitriles

Some major transformation related biological and growth properties have been analysed in an in vitro, system to characterize the PMM-14 cell line, a 20-methylcholanthrene induced transformed mouse embryonic fibroblast cell line. The population doubling time of this cell line was 19 h with moderately high saturation density and plating efficiency. Attachment detachment properties showed reduced adhesion to substratum. Cytogenetic study revealed the existence of a large number of Robertsonian biarmed chromosomes with hypertriploid modal range (60-69) represented by about 21% cells. These cells showed enhanced concanavalin A agglutinability which was reduced by trypsin treatment. Its neoplastic nature was finally established by its ability to grow in agar with a high plating efficiency.

Topics: Agar; Animals; Cell Adhesion; Cell Aggregation; Cell Cycle; Cell Line; Cell Transformation, Neoplastic; Concanavalin A; Culture Media; Fibroblasts; In Vitro Techniques; Karyotyping; Methylcholanthrene; Mice; Trypsin

Polypeptide growth factor activity in serum can be destroyed by treatment with dithiothreitol. When such growth-factor-inactivated serum is used as a supplement of culture media instead of regular serum, normal rat kidney (NRK) cells become quiescent unless defined polypeptide growth factors like insulin and epidermal growth factor (EGF) are added. On this basis a growth-factor-defined medium has been developed for NRK cells, which permits cell proliferation as rapidly as in media supplemented with serum, even at low cell densities. Moreover, cells can be serially passaged in this medium. NRK cells can be induced to grow in semisolid media when incubated with transforming growth factors. The growth-factor-defined medium permits soft agar growth experiments of NRK cells, without interference from polypeptide growth factors in serum. Using this assay system we have shown that EGF alone is unable to induce any degree of anchorage-independent growth in NRK cells. However, a recently identified transforming growth factor from mouse neuroblastoma cells which does not compete with EGF for receptor binding is able to induce progressively growing colonies of NRK cells in soft agar, even without additional EGF.

Topics: Agar; Animals; Blood; Cell Division; Cell Line; Cell Transformation, Neoplastic; Clone Cells; Culture Media; Epidermal Growth Factor; ErbB Receptors; Growth Substances; Kidney; Mice; Neuroblastoma; Peptides; Phenotype; Rats; Receptors, Cell Surface; Transforming Growth Factors

Topics: Agar; Blood; Carcinogens; Cell Transformation, Neoplastic; Culture Media; Diploidy; Fibroblasts; Genetic Markers; Humans; Peptides; Time Factors; Transforming Growth Factors

Cells from a cloned line of spontaneously transformed 3T3 cells had a colony-forming efficiency in agar (CFEag) of about 10-15% and induced poorly differentiated sarcomas when injected into nude mice. These tumor cells were recultured in vitro and tested for their ability to grow in suspension. Initially, the tumor cells had a CFEag which was a hundredfold to a thousandfold lower than the cells that had been grown only in vitro. After 5-8 further weekly passages, however, the tumor lines recovered their original ability to grow in agar. For determination as to whether this increased CFEag was due to selection of cells with a higher CFEag from the tumor cell population or to adaptation of many of the tumor cells to agar growth, clones were isolated directly from a primary tumor, and each was tested weekly for agar colony formation. All of the tumor clones, as well as the uncloned tumor line, were able to recover their original ability to grow in agar. However, one tumor clone had a relatively high CFEag in the first assay, so the selection hypothesis could not be totally excluded. The initial low CFEag of the recultured cells was not due to the presence of normal nude mouse cells in the population. Before in vivo growth no clones could be isolated from the sublines that had as low a CFEag as the tumor cells isolated after in vivo growth. Tumor cells that had been repeatedly passaged in vivo still had a much lower CFEag than the input cells upon explantation into culture. The results suggest that phenotypic alterations observed during tumor growth and subsequent cultivation have an epigenetic basis.

Topics: Agar; Animals; Animals, Suckling; Cell Division; Cell Survival; Cell Transformation, Neoplastic; Clone Cells; DNA; Histological Techniques; Mice; Mice, Nude; Neoplasm Transplantation; Neoplasms; Tumor Stem Cell Assay

Hamster embryo cells were transformed by African green monkey lymphotropic papovavirus (LPV). The transformed cells contained intranuclear T-antigens demonstrable by fluorescent antibody staining with hamster anti-LPV serum. Analysis of uncloned and cloned lines of transformed cells for LPV sequences revealed that the viral DNA was present as free nonintegrated and integrated genomes; there were approximately 10 copies of free DNA and about one to two copies of integrated genomes per cell. The cells were highly tumorigenic when inoculated into hamsters and produced progressively growing tumors in 100% of newborn or 10-day-old hamsters that were inoculated with LPV-transformed cells. The serum from tumor-bearing hamsters reacted with LPV-transformed cells and also showed a weak reaction with simian virus 40-, BK virus-, and JC virus-transformed cells, thereby showing an antigenic relationship with the T-antigens of other primate polyomaviruses. The large T-antigen of LPV was found to be an 84,000-molecular-weight protein which was immunoprecipitated by hamster anti-LPV (antiviral) as well as by tumor serum.

Topics: Agar; Animals; Antigens, Viral, Tumor; Blood; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Clone Cells; Cricetinae; Culture Media; DNA, Viral; Embryo, Mammalian; Neoplasms, Experimental; Polyomavirus; Tumor Virus Infections

Rat 3Y1 cells were transfected with recombinant gARC ( pSV2gpt carrying the adenovirus 12 early region 1 [E1] gene), and focus formation was observed in monolayer cultures after culture of cells in gpt-selective medium (Eagle medium containing 10% fetal calf serum, xanthine, thymidine, aminopterin, and mycophenolic acid) for 10 days, followed by focus formation. Transformed E1Y cell lines were then established from these foci. The E1Y cells were transformed morphologically similarly to cells transformed with intact adenovirus 12 DNA but formed no colonies in soft-agar culture and induced tumors in transplanted rats only after a long incubation period. For the establishment of completely transformed cells, 3Y1 cells were transformed with combinations of gARC , pE3 (pBR322 carrying the adenovirus 12 E3 gene), and gE4 ( pSV2gpt carrying the adenovirus 12 E4 gene) DNA. E1- 3Y cells (3Y1 cells transformed with gARC and pE3 DNA), E1- 4Y cells (3Y1 cells transformed with gARC and gE4 DNA), and E1-3- 4Y cells (3Y1 cells transformed with gARC , pE3 , and gE4 DNA) were established. These transformed cell lines were compared for growth in Eagle medium with 2 or 10% fetal calf serum, colony formation in soft-agar culture, and tumor growth in rats transplanted with the transformed cells. Several transformed cell lines of E1- 4Y and E1-3- 4Y cells showed colony formation in soft-agar culture and abundant expression of the E1B gene. T antigen f was seen by immunofluorescence as flecks in these cells, in which the E4 gene was transcribed, but was not seen in E1Y cells, suggesting that T antigen f was encoded by the E4 gene. The suggestion was confirmed by the observation that T antigen f was detected in COS-1 cells transfected singly with gE4 DNA by immunofluorescence with polyclonal and monoclonal antibodies. Transcription of the E4 gene was confirmed in gE4 -transfected COS-1 cells. T antigen f, one of the E4 gene products, was identified as a polypeptide of molecular weight 11,000 (E4- 11K ) by immunoprecipitation with monoclonal antibodies. The above results also suggest that expression of the E4 gene gives cells the advantage of forming colonies in soft-agar culture. A tendency was noticed for E1B gene expression to be enhanced by E4 gene expression. The relationship between enhancement of colony formation in soft-agar culture and enhancement of E1B gene expression is discussed.

Topics: Adenoviruses, Human; Agar; Animals; Base Sequence; Cell Line; Cell Transformation, Neoplastic; Chlorocebus aethiops; Cloning, Molecular; DNA Restriction Enzymes; DNA, Recombinant; Embryo, Mammalian; Genes, Viral; Humans; Rats; Rats, Inbred F344; Species Specificity; Transfection

The relationship of cytogenetic changes with the acquisition of an indefinite life span in vitro, the ability of cells to grow in soft agar and their tumourigenicity in syngeneic animals has been studied in control, trans-7,8-dihydrodiolbenzo(a)pyrene and 7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)-pyrene-treated secondary cultures derived from Chinese hamster embryonic lung. Karyotype analysis revealed a sequence of chromosome changes as the cells progressed through culture. Aneuploidy, namely trisomy of chromosome 4, the long arm in particular, was an early dominant change. The possible association of this trisomy with the acquisition of immortality in vitro is implicated, although the involvement of other nonrandom chromosome changes cannot be eliminated, implying that there may be several genomic sites in the Chinese hamster which may potentially be involved with the acquisition of unlimited growth potential. Neither the ability of cells to grow in soft agar nor as tumours could be associated with any specific chromosome(s). Double minutes were observed in metaphases from the cell lines, agar colonies and tumours; their possible relationship with growth advantage is discussed.

Topics: 7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; Agar; Animals; Benzopyrenes; Carcinogens; Cell Division; Cell Line; Cell Survival; Cell Transformation, Neoplastic; Cells, Cultured; Chromosomes; Cricetinae; Cricetulus; Dihydroxydihydrobenzopyrenes; Fetus; Karyotyping; Lung; Sarcoma, Experimental

A subline of cloned spontaneously transformed BALB/3T3 cells had a colony-forming efficiency (CFE) in agar of 5 to 20%. Individual agar colonies isolated and reseeded into agar were not significantly more efficient at initiating colonies than the original unselected subline. Four successive cycles of agar growth and selection also failed to increase the mean CFE in agar. Randomly selected clones isolated on a plastic surface all had the capacity to grow in agar. These results suggest that the failure of the majority of the cells to grow in agar is not the result of an intrinsic or heritable inability to do so. The ability to initiate a colony in agar seems to vary phenotypically from cell to cell. In contrast, agar colonies isolated from some tumor cell lines (originating from related spontaneously transformed 3T3 cells) and reseeded in agar had a higher CFE than the unselected tumor cell lines. In one case, this increased CFE in agar was lost when the cells were passaged on plastic without further selection for agar growth. Thus, expression of the anchorage-independent phenotype may vary, even among related cloned populations of transformed cells.

Topics: Agar; Animals; Cell Adhesion; Cell Cycle; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Clone Cells; Mice; Mice, Inbred BALB C; Phenotype

The differentiation pattern of two human germ cell tumors, grown in nude mice and in vitro is described. Tumor A was an embryonal carcinoma (EC) of borderline histology with characteristics of yolk sac tumor and of seminoma; tumor B was a teratocarcinoma with yolk sac elements and syncytiotrophoblastic giant cells. The morphology of an EC as well as cytogenetic characteristics were maintained during 20 passages in nude mice from tumor A and over 11 passages from tumor B. Tumor A did not grow in vitro. Cell suspensions prepared from xenografted tumor B grew into cystic embryoid bodies in semi-solid tissue culture medium. These embryoid bodies showed cuboidal and flattened cells with microvilli, junctional complexes, peripheral microfilaments, and annulated lamellae, reminiscent of the 'inner cell mass' of a blastula and of endoderm, respectively. When such colonies were transplanted into nude mice, however, only tumors with the morphology found in the transplants appeared.

Topics: Agar; Animals; Cell Transformation, Neoplastic; Cells, Cultured; Humans; Male; Mice; Mice, Nude; Microscopy, Electron; Microscopy, Electron, Scanning; Neoplasm Transplantation; Teratoma; Testicular Neoplasms

Stromal cell cultures obtained from human endometrium were treated repetitively with N-methyl-N'-nitro-N-nitrosoguanidine in vitro at concentrations ranging from 0.5 to 4.0 micrograms/ml, and alterations in growth potential and morphology were analyzed. A single exposure to the carcinogen resulted in morphological evidence of toxicity and reductions in growth rates, plating efficiency, and saturation density as compared to solvent-treated control cells. Cytotoxicity was reduced after additional exposures to the carcinogen. Following repetitive treatments with N-methyl-N'-nitro-N-nitrosoguanidine, human endometrial stromal cells developed enhanced growth potential, the capacity to form macroscopic colonies in soft agar, and elevated gamma-glutamyltranspeptidase activity. Carcinogen-treated cells displayed atypical morphology characterized by irregularities in cell and nuclear size and shape, large bizarre nucleoli, increased nuclear:cytoplasmic ratios, and cellular crowding. Control cells did not display altered morphology or growth parameters even following multiple exposures to solvent and repetitive subculturing. These alterations in growth potential and morphology suggest that the cells are progressing towards preneoplastic and perhaps neoplastic transformation in vitro.

Topics: Agar; Cell Aggregation; Cell Division; Cell Line; Cell Nucleolus; Cell Nucleus; Cell Transformation, Neoplastic; Cytoplasm; Endometrium; Female; gamma-Glutamyltransferase; Humans; In Vitro Techniques; Methylnitronitrosoguanidine; Time Factors

Inbred X/Gf mice are reported to have a very low incidence of spontaneous and induced tumors. Experiments were performed to determine whether the cells of the X/Gf mouse are themselves resistant to in vitro oncogenesis or the organism as a whole is surveyed by a homeostatic mechanism that prevents the expression of potential neoplasia. Cultures of X/Gf or C57BL/6J embryonic fibroblasts were exposed to the chemical carcinogens 7,12-dimethylbenz[a]anthracene and benzo[a]pyrene to test whether the cells of the X/Gf mouse are themselves resistant to oncogenesis. The chemical carcinogens caused transformation of both X/Gf and C57BL/6 fibroblasts into cells that demonstrated altered growth patterns in liquid culture and grew in a clonal fashion in soft agar. Cloned transformed cells were shown to be malignant and resulted in the growth of sarcomas in vivo. X/Gf mice were decidedly more resistant than were C57BL/6 mice to their respective clones of transformed cells. The resistance to neoplastic growth demonstrated by the X/Gf mice was inherited by (C57BL/6J X X/Gf)F1 mice that were resistant to the growth of X/Gf-transformed and C57BL/6-transformed cells at cell doses at which the parental X/Gf mice had survived but at which the C57BL/6 had succumbed to progressively growing tumors. It has thus been established that the tumor resistance of the X/Gf mice is not based on the resistance of isolated cells from these animals to malignant transformation, but rather is attributable to an in vivo homeostatic mechanism that prevents tumor growth.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Agar; Animals; Benz(a)Anthracenes; Benzo(a)pyrene; Benzopyrenes; Cell Transformation, Neoplastic; Cells, Cultured; Embryo, Mammalian; Female; Male; Mice; Mice, Inbred Strains; Neoplasm Transplantation; Sarcoma, Experimental

Formation of morphologically transformed colonies and the ability to grow in semi-solid agar has been compared for 3 different cell lines from hamster embryo and for primary hamster embryo cells. By manipulating the growth conditions, transformed colony morphology and growth in agar could be induced for all cell types studied. Conditions that induced morphologically transformed colonies, also produced growing colonies in agar. One cell line and the primary cells needed the presence of the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate for the expression of transformed morphology and agar growth, while the two other cell lines produced both morphologically transformed colonies and growth in soft agar without any additions. None of the cell lines would produce morphologically transformed colonies in the presence of newborn bovine serum. Likewise, the cells were dependent on fetal bovine serum in order to grow in soft agar, except for one of the cell lines which produced a low number of agar growing colonies in newborn bovine serum. The data indicate a close relation between morphological transformation and growth in soft agar.

Topics: Agar; Animals; Benzo(a)pyrene; Benzopyrenes; Cell Adhesion; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cricetinae; Culture Media; Embryo, Mammalian; Neoplasms, Experimental; Teratoma; Tetradecanoylphorbol Acetate

A nontransformed clone and a spontaneously transformed clone were isolated from a twice-recloned line of Balb/3T3 cells. At different times two sublines were initiated from the nontransformed clone, and three were initiated from the transformed clone. The sublines were maintained in parallel passages under the same conditions. Each subline was distinctive in appearance and fell into the same rank order in a variety of growth parameters in vitro. Colony formation in agar and tumor formation in mice occurred only in the morphologically transformed sublines, but there was no quantitative correlation between the two properties or with the rate of glucose utilization. Two of the cell populations derived from noninbred NIH nude mouse tumors of the 3 transformed sublines differed in agar colony formation from the parental sublines. The results indicate that there is an immense capacity for variation in cultured animal cells involving many unrelated characteristics expressed in a way that is difficult, if not impossible, to explain by conventional genetic models.

Topics: Agar; Animals; Cell Cycle; Cell Transformation, Neoplastic; Cells, Cultured; Clone Cells; Culture Media; Glucose; Hydrogen-Ion Concentration; Kinetics; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation

Established cell lines from 8 human squamous cell carcinomas (SCC) together with normal human keratinocytes, have been investigated for their ability to grow in soft agar and as xenografts when injected as a single cell suspension into immunologically incompetent mice. One of 8 SCC lines formed colonies with efficiencies greater than 1% in soft agar, and only 2 formed progressively growing tumors when injected into animals. It is concluded that these 2 criteria are not reliable markers of malignant transformation in squamous epithelia unless cytological criteria are also applied.

Topics: Agar; Animals; Carcinoma, Squamous Cell; Cell Line; Cell Transformation, Neoplastic; Cells, Cultured; Humans; Immunocompetence; Mice; Mice, Nude; Neoplasm Transplantation; Skin

Human carcinoma tissues were grown in culture for two to four weeks using the two-layer soft agar technique. All cultures that showed growth of tumor cell colonies also showed well-preserved, apparently healthy tumor cells lying singly. These cells showed neither proliferative capacity nor necrosis or morphologic degeneration during the time in soft agar. Thus, morphologic criteria seem to be poor indicators of tumor cell proliferative potential, at least in the short term. However, the method of soft agar tumor clonogenic cell culture itself provided a direct measure of tumor cell proliferative capacity, ie, the formation of colonies from single tumor cells. This may be valuable in directly assessing the presence of "viable" tumor cells in biopsy specimens taken after therapy, and thus guide further patient therapy.

Topics: Agar; Aged; Carcinoma; Cell Transformation, Neoplastic; Cells, Cultured; Female; Humans; Male

As part of an attempt to reproduce Styles' cell transformation assay, the effect of serum concentration on the growth of normal and 4-nitroquinoline-1-oxide (4-NQO)-treated BHK 21 Cl 13 cells in soft agar medium was examined. Using medium with 10% newborn calf serum, dilution of control cultures from 5 x 10(4) to 1.56 x 10(3) cells/ml caused little increase in the number of countable (greater than 0.3 mm diameter) colonies, but 4NQO caused a marked dose-related increase. In contrast, using 20% of the same batch of serum, 4NQO-treated groups and controls diluted to comparable viability counts showed very similar increases in countable colonies. Clones greater than 0.3 mm diameter isolated from control cultures with 20% serum did not appear to be transformed when grown in agar with 10% serum. These data indicate that factors other than neoplastic transformation can influence the growth of BHK 21 Cl 13 cells in soft agar medium.

Topics: 4-Nitroquinoline-1-oxide; Agar; Animals; Cell Line; Cell Transformation, Neoplastic; Cricetinae; Culture Media; Kidney; Mesocricetus

T lymphocytic colony formation by peripheral lymphocytes separated by discontinuous albumin gradient centrifugation was evaluated in 8 patients with Philadelphia (Ph1)-positive chronic myeloid leukemia (CML). Colonies were obtained using a liquid-on-agar culture system recently introduced (PHA overlayer-leukocyte feeder layer assay) which has been shown to be simple and reliable. The pattern of colony growth in CML and in normal controls was similar, the peak ranging from the 4th to the 6th day. Also the morphological aspects of colonies did not differ in the two groups. Cells recovered from CML lymphocytic colonies were shown to belong to T cell lineage, as they are able to form spontaneous E-rosettes and to respond to mitogenic stimulation in vitro. In contrast, cells recovered from all other cultured fractions failed to display these properties. Cytogenetic analysis showed that T colony cells were Ph1-negative whereas the chromosome anomaly was found in nonlymphoid colonies of the same patients, thus suggesting a nonclonal origin of T lymphocytes in CML.

Topics: Agar; Cell Transformation, Neoplastic; Cells, Cultured; Centrifugation, Density Gradient; Chromosomes, Human, 21-22 and Y; Concanavalin A; Humans; Leukemia, Myeloid; Phytohemagglutinins; Pokeweed Mitogens; Rosette Formation; T-Lymphocytes

The growth characteristics of LT-2 cells, an epithelial squamous cell carcinoma, clearly separate the phenotypes of anchorage-independent growth in soft agar and tumor formation in vivo. LT-2 readily grows and forms tumor nodules in the nude mouse but does not proliferate anchorage independently in soft agar. This distinction is confirmed by the observation that cells explanted from nude mouse tumor nodules and cultured in vitro still do not clone in soft agar. The human origin of tumor nodule cells was confirmed by karyotyping. LT-2 cells have an aneuploid karyotype which has persisted for 4.5 years in culture with considerable variation in chromosomal number. Passage through the nude mouse did not select for any "tumor" clone since marked chromosomal variation was still noted by cells explanted from tumor nodules. Tumor cells formed a well-differentiated skin with keratin formation in the nude mouse despite wide karotypic variations of cells and years of in vitro culture. Strict monolayer growth was noted by LT-2 cells when grown in culture flasks and also by cells explanted from tumor nodules, indicating that monolayer growth and nude mouse tumorigenicity are also separate phenotypes.

Topics: Agar; Aneuploidy; Animals; Carcinoma, Squamous Cell; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cytological Techniques; Karyotyping; Mice; Mice, Nude; Neoplasm Transplantation; Neoplasms, Experimental; Phenotype

In vitro assays that permit cloning of tumour cells in soft agar have been improved during the last 5 years. Two of them (2, 10) are claimed to be useful as test systems for the screening of new anticancer drugs and even for drug sensitivity testing of individual human tumours in the devicing of individualized cancer chemotherapy regimens. Three assays were investigated for this report: those of Toshio Kuroki (TK) (11) and Hamburger and Salmon (HS) (5, 10) and that in use for bone marrow cell cultures (BM) (3). Cells of various origins were tested for their growth capacity and colony formation in these three assays. Included were cells of 10 established lines classified as malignant or nonmalignant according to the in vivo malignancy test (6). Cells freshly derived from two tumours ans those from five tumours after 2-10 passages in monolayer culture were also used as test cells. The BM assay gave the best results. Up to now, a 100 per cent correlation has been found between the in vivo and in vitro test. Investigations are under way to determine whether this assay can also be used as a transformation assay using cells with a low transformation rate.

Topics: Agar; Animals; Cell Line; Cell Transformation, Neoplastic; Colony-Forming Units Assay; Culture Media; Humans; Mice; Rats

Using a specific stain and electron microscopy, small numbers of mast cells were detected in human bone marrow cultures. However, they were not detected in agar cultures containing murine bone marrow cells. In bone marrow cultures from three patients with acute myeloid leukemia the number of mast cells was elevated.

Topics: Agar; Animals; Bone Marrow Cells; Cell Transformation, Neoplastic; Cells, Cultured; Cytoplasmic Granules; Humans; Leukemia, Myeloid; Mast Cells; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Staining and Labeling

Simian virus 40 tsA-transformed BALB/c-3T3 cells isolated as foci of overgrowth in liquid medium were compared with those isolated as colonies in soft agar. Efficiencies of transformation were equivalent in the two procedures. Cells isolated as foci were able to grow in agar and vice versa. No difference in temperature sensitivity of the transformed phenotype was detected when tsA transformants selected in agar were compared with those selected as foci. The use of the two different transformation procedures, then, did not form the basis for generation of different transformed phenotypes, and transformants generated in both ways were dependent upon expression of the A gene for maintenance of the transformed state.

Topics: Agar; Animals; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Culture Media; Fibroblasts; Mice; Mice, Inbred BALB C; Mutation; Phenotype; Simian virus 40; Temperature; Virus Replication

Infection of long-term BDF1 marrow cultures with Friend leukaemia virus complex (FLV) induced transformed cells with myelomonocytic characteristics, which were isolated only 14 days after the viral infection. Criteria for transformation were growth in suspension cultures and high plating efficiency in agar. The lymphatic leukaemia virus (LLV) replicates in these suspension cultures, but the spleen focus-forming virus (SFFV) component of the FLV complex has not been detected. Injection of the transformed cells into syngeneic neonatal or adult mice leads to the development of leukaemia which can be demonstrated to be of donor origin by the presence of two metacentric marker chromosomes which are also seen in the cultured cells.

Topics: Agar; Animals; Bone Marrow; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Colony-Forming Units Assay; Friend murine leukemia virus; Genetic Markers; Leukemia, Experimental; Leukemia, Myeloid; Mice; Neoplasm Transplantation; Time Factors; Transplantation, Isogeneic

Topics: Agar; Animals; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cricetinae; Culture Media; Fibrinolysis; Neoplasms, Experimental; Plasminogen Activators

To examine the role of germinal mutation in transformation by phorbol esters, we studied the induction of anchorage-independent variants of mutant human diploid fibroblasts derived from normal-appearing skin of individuals with hereditary adenomatosis of the colon and rectum (ACR). Liquid cultures were chronically exposed to 12-0-tetradecanoyl phorbol-13-acetate (TPA), then plated in agar and injected subcutaneously into athymic mice. Cultured ACR cells showed an unusual biphasic dose response to TPA. Colony-forming cells in agar were obtained at a frequency of about 5 x 10(-5). They did not, however, seem to increase in frequency during subsequent passages in liquid cultures continuously exposed to TPA. The isolated anchorage-transformed clones showed an altered clonal morphology and a considerable increase in cloning efficiency in liquid cultures and agar. The results suggest that ACR cells may be used to screen for potential tumor promoters in our environment.

Topics: Adenoma; Agar; Animals; Cell Transformation, Neoplastic; Cells, Cultured; Colonic Neoplasms; Fibroblasts; Humans; Mice; Mice, Nude; Neoplasm Transplantation; Phorbols; Rectal Neoplasms; Skin; Tetradecanoylphorbol Acetate

Cultures derived from rat brains at different times during the latent period of brain-tumour induction by N-ethyl-N-nitrosourea (ENU) showed increased plasminogen activator (PA) activity before being able to form colonies in agar. Control cultures from buffer-exposed animals showed neither property at comparable passages. More detailed investigations, using a culture derived from foetal brains only 2 days after exposure to ENU and clones from this culture, showed a sequence of low PA activity, then increased activity, followed by the ability to form colonies in agar, suggesting progressive transformation of cells in culture. Continuous culturing in the presence of the mouse skin tumour promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), did not accelerate the rate at which these two properties were acquired, but did cause a much greater increase of PA activity once this started to rise. If included in the assay mixture TPA also increased the PA activity of the cells. It therefore appears that in this system TPA can modulate PA activity under certain circumstances.

Topics: Agar; Animals; Brain; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Ethylnitrosourea; Phorbols; Plasminogen Activators; Rats; Tetradecanoylphorbol Acetate

The characteristics of WI-38 CT-1 cells, which had been transformed by treatment with Co-60 gamma rays, were compared with those of the control WI-38 cells in order to identify parameters of malignant transformation in cultures of normal human diploid cells. The transformed WI-38 CT-1 cells were different from the control WI-38 cells in the following respects: (1) epithelial-like morphology of the cells, (2) reduced serum requirement for cell proliferation, (3) increased colony formation in soft agar, (4) increased saturation density, and (5) resistance to the cytostatic effect of theophylline on cell proliferation. On the other hand, no appreciable difference was detected between the control and the transformed cells in (1) population doubling time and (2) sensitivity to Co-60 gamma rays.

Topics: Agar; Animals; Cattle; Cell Transformation, Neoplastic; Cobalt Radioisotopes; Colony-Forming Units Assay; Cricetinae; Culture Media; Humans; Mice; Mitosis; Rats; Theophylline

The growth and differentiation of granulopoietic progenitor cells from 15 patients with haemopoietic dysplasia were studied by in vitro culture in agar-gel. After 14 d in culture whole colonies and clusters were transferred to glass slides and were stained with a modified Papanicolaou technique. The preparations were examined for cellular differentiation by counting the number of mature cells (band forms and polymorphonuclear cells) and a mitotic index was calculated from the number of mitotic cells. Patients with defective colony formation showed granulopoietic maturation defects and a reduced mitotic index was found in some colonies. Patients who had colony counts within the normal range, however, showed normal in vitro maturation. Defective colony growth in haemopoietic dysplasia generally indicates a malignant course and can occasionally be related to leukaemic transformation. The finding of in vitro maturation defects in an additional culture abnormality which may indicate a deteriorating clinical course. A defective maturation and a reduced mitotic index in vitro add support to the concept of clonal progression in malignant haemopoietic dysplasia.

Topics: Adult; Agar; Aged; Bone Marrow; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Female; Humans; Male; Middle Aged; Mitotic Index; Myeloproliferative Disorders; Preleukemia

The effects of certain factors on the performance of the cell transformation test of Styles were examined by testing the demonstrability of transformation of cells of a subclone of BHK21 C13 in response to treatment with 4-nitroquinoline-1-oxide, N-methyl-N'-nitro-N-nitrosoguanidine, benzo[a]pyrene and 2-acetamidofluorene. An important requirement for success was supplementation of the soft agar medium with a serum which supported microcolony formation by a high proportion of the cells. Increasing the concentration of an unsuitable serum improved the results obtained; this suggests that the serum was inadequate rather than inhibitory. Alteration of the concentration of S-9 fraction used to activate the precarcinogens benzo[a]pyrene and 2-acetamidofluorene had little effect on the induction of transformation by either. A clearer distinction between the transformation frequencies for control and treated cells was usually obtained when 500 microm rather than 200 microm was the minimum diameter set for definition of transformation. It is suggested that, to assess the validity of results obtained with compounds which appear to induce transformation only at highly toxic levels, transformation frequencies for treated cells should be compared with those for control cells seeded at comparable densities.

Topics: 2-Acetylaminofluorene; 4-Nitroquinoline-1-oxide; Agar; Animals; Benzo(a)pyrene; Biotransformation; Blood Physiological Phenomena; Carcinogens; Cell Count; Cell Line, Transformed; Cell Transformation, Neoplastic; Cricetinae; Culture Media; Dose-Response Relationship, Drug; Drug Synergism; Fibroblasts; Mesocricetus; Methylnitronitrosoguanidine; Microsomes, Liver; Rats

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

The transplacental host-mediated hamster cell culture system was used to test a variety of solvents and chemicals of unknown and known (positive and negative) activity for their ability to induce morphologic transformation of cells and growth in agar. Examination of approximately 13,000 colonies of cells from untreated animals yielded no transformants, thus demonstrating no spontaneous transformation in the system. Similar negative results were obtained after animals were treated with the solvents acetone, ethanol, dimethyl sulfoxide, dimethylformamide, and trioctanoin oil. Several known carcinogens, including benzo[a]pyrene, methylnitrosourethane, urethan, and diethylnitrosamine, were positive for transforming activity. Three pesticides, carbaryl, methomyl, and landrin, and their N-nitroso derivatives were tested. All the nitrosated forms had transforming activity, but only one of the pesticides, landrin, was positive. In all experiments conducted, results of the agar-growth test correlated well with tests for morphologic transformation. The transplacental hamster embryol cell culture system therefore detected transforming activity of N-nitroso compounds and some known carcinogens.

Topics: Agar; Animals; Carcinogens; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Cricetinae; Drug Evaluation, Preclinical; Embryo, Mammalian; Female; Maternal-Fetal Exchange; Nitroso Compounds; Pesticides; Pregnancy

Transformation of primary hamster embryo cells was investigated using 3-methylcholanthrene (MCA), a combination of MCA and 12-O-tetradecanoylphorbol-13-acetate (TPA), and initiation with MCA or dibenz(a,h)anthracene (DBA) followed by promotion with TPA. Evidence for transformation was (a) abnormal cellular morphology, (b) increased lifespan, (c) growth in soft agar, and (d) tumour induction by s.c. inoculation into suckling hamsters.Cells treated with either MCA or MCA+TPA showed the same latent period to morphological transformation, although their tumorigenic potential varied. Cells did not form tumours when TPA was administered 7 days after treatment with either MCA or DBA. However, when administration of TPA was delayed to 27 days after treatment with a transforming dose of MCA or a subthreshold dose of DBA, the cells transformed and produced tumours in hamsters.Our results show that TPA may act as an inhibitor or promoter, depending on the length of time between treatment of the hamster embryo cells with the carcinogen and administration of the TPA. It appears that treatment of cells with TPA before the initiating event is complete inhibits or delays the development of their ability to induce tumours in animals or grow in soft agar. However, with a sufficient interval between the application of the initiating carcinogen and the promoter, transformation occurs, and the ability of cells treated with subthreshold doses of DBA to form tumours is enhanced.

Topics: Agar; Animals; Benz(a)Anthracenes; Cell Transformation, Neoplastic; Cricetinae; Methylcholanthrene; Neoplasm Transplantation; Neoplasms, Experimental; Phorbols; Tetradecanoylphorbol Acetate; Time Factors

Topics: Agar; Animals; Carcinogens; Cell Line; Cell Survival; Cell Transformation, Neoplastic; Cells, Cultured; Culture Media; Drug Evaluation, Preclinical; Female; Leukemia, Experimental; Pregnancy; Rats; Rauscher Virus

A simian virus 40 (SV40)-transformed line of human parapharyngeal cells (SV-TGo) was cultured in semisolid agar to determine its ability to grow in the absence of an anchoring substratum and to evaluate any phenotypic changes that might have resulted during the isolation of sublines specifically selected for anchorage independence. After 2--3 weeks and 14--15 population doublings in culture, SV-TGo plated with over 1,000% higher efficiency than negative controls (F2408 cells). Sublines, 0.3--2.0 mm in diameter, were isolated and transferred to Leighton tubes in which they underwent an additional 0--7 divisions before senescence after 39--44 total population doublings. Subline phenotype was identical to the original parental phenotype, including epithelioid morphology, organized pattern of growth, extreme sensitivity to density-dependent inhibition of growth, and continuous production of infectious SV40 as detected by the combined tests of cocultivation and direct isolation. Limited division potential was within the range observed for the parental line. The ability to grow in agar without identifiable phenotypic changes was therefore confirmed for this line of SV40-transformed human epithelioid cells.

Topics: Agar; Cell Adhesion; Cell Line; Cell Transformation, Neoplastic; Clone Cells; Epithelial Cells; Humans; Nasopharynx; Phenotype; Simian virus 40; Virus Replication

Fisher rat fibroblasts (FR 3T3), transformed with the tsA30 mutant of simian virus 40 and selected by colony formation in soft agar, maintained the transformed phenotype at high temperature, whereas most transformants isolated from foci were found to undergo a phenotypic reversion toward the normal state in their saturation density, ability to grow in soft agar, and rate of 2-deoxyglucose transport. The temperature-independent phenotype observed in agar-selected transformants was not due to a reversion of the viral mutation. These results, similar to those previously obtained with polyoma virus tsa mutants, further suggest that two distinct mechanisms may operate in both cases for maintaining the transformed phenotype. Immunofluorescence studies suggested a different regulation of T antigen synthesis in these two classes of transformants.

Topics: Agar; Animals; Antigens, Viral; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Deoxyglucose; Mutation; Phenotype; Rats; Simian virus 40; Temperature; Virus Replication

Topics: Agar; Animals; Avian Sarcoma Viruses; Caseins; Cell Transformation, Neoplastic; Cell Transformation, Viral; Chick Embryo; Culture Techniques; Fibroblasts; Genes, Viral; Plasminogen Activators; Virus Replication

Infection of normal rat fibroblasts (FR 3T3) with the early tsa mutant of polyoma virus may lead to either the A or the N phenotype, tsa-A transformants, originally derived by agar selection, are not temperature dependent for maintenance of the transformed phenotype, whereas tsa-N transormants revert at high temperature to normal growth control. A transformants did not result from an independent cellular mutation selected in agar medium, but rather from a transformation process distinct from that leading to the N state. It occurred in both liquid and agar media when the infected cells were maintained under growth-restricting conditions, such as absence of anchorage and contact inhibition at confluency. N transformation occurred in cells maintained in active growth after virus infection (sparse cultures on a solid substratum). Physiological conditions during a critical period after virus infection thus appear to be a crucial parameter of the transformation process.

Topics: Agar; Animals; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Clone Cells; Fibroblasts; Mutation; Phenotype; Polyomavirus; Rats; Temperature

A human revertant cell line, derived from non-producer human osteosarcoma cells (NP/KHOS) induced by Kirsten murine sarcoma virus, was treated in vitro with various levels of polycyclic aromatic hydrocarbons (3-methylcholanthrene, 7,12-dimethylbenz(alpha)anthracene, and benzo(alpha)pyrene [BP] or dimethyl sulfoxide (control). Cells treated only with 3-methylcholanthrene and 7,12-dimethylbenz(alpha)anthracene underwent morphological alteration in vitro, and produced tumors when injected into NIH nude mice. These human revertant cells may be a useful in vitro tool in screening for the potential chemical carcinogens in human cell systems.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Agar; Benzopyrenes; Carcinogens; Cell Aggregation; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cytotoxicity Tests, Immunologic; Humans; Methylcholanthrene; Polycyclic Compounds

Topics: 9,10-Dimethyl-1,2-benzanthracene; Agar; Animals; Benz(a)Anthracenes; Cell Line; Cell Transformation, Neoplastic; Culture Media; Neoplasm Transplantation; Neoplasms, Experimental; Rats; Time Factors; Tracheal Neoplasms; Transplantation, Isogeneic

Topics: Agar; Animals; Cell Adhesion; Cell Division; Cell Transformation, Neoplastic; Clone Cells; Methylnitronitrosoguanidine; Mice; Mice, Inbred BALB C; Neoplasm Transplantation; Neoplasms, Experimental; Skin Neoplasms; Tetradecanoylphorbol Acetate; Transplantation, Isogeneic

Primary rabbit kidney cells were transformed by BKV(MM), a papovavirus isolated from the urine of a child with the Wiskott-Aldrich syndrome. The transformed cells contained BK T-antigen, but no antigen that reacted with SV40 U-antiserum. The transformed cells failed to produce tumors in nude mice, and BKV (MM) was not rescued from transformed cells by cell fusion or chemical induction methods. The transformed cells supported the growth of rabbit kidney vacuolating virus (RKV), and could be used to quantitate RKV by plaque formation under an agar overlay.

Topics: Agar; Animals; Antigens, Viral; BK Virus; Cell Aggregation; Cell Line; Cell Transformation, Neoplastic; Cells, Cultured; Child; Fluorescent Antibody Technique; Humans; Kidney; Mice; Polyomavirus; Rabbits; Viral Plaque Assay; Wiskott-Aldrich Syndrome

Growth behavior in vitro of 3T3 cells and the cells transformed by polyoma or SV40 virus was studied after their treatment with dextran sulfate. The transformed cells treated with dextran sulfated showed decreased saturation density compared with untreated cells, whereas saturation density of 3T3 cells was hardly affected by treatment with dextran sulfate. The ratio of saturation density of untreated SV3T3 cells to that of the treated cells was 64.1% at 5 microng/ml and was 43.4% at 10 microng/ml of dextran sulfate. In Py3T3 cells the ratio was 45.0% at both concentrations of 10 and 20 microng/ml. Plating efficiency was not affected in 3 cell lines tested after treatment with dextran sulfate. The treated cells showed a tendency to form thin and not piled-up colonies in the central area. The treated SV3T3 and Py3T3 cells were flattened in shape compared with the untreated cells. In agar medium the treated SU 3T3 and Py 3T3 cells showed the tendency to stop growing after forming small colonies, whereas the untreated cells kept growing anf formed large colonies. These results suggested that dextran sulfate altered temporarily the growth of the transformed cells to that of untransformed cells.

Topics: Agar; Cell Division; Cell Line; Cell Transformation, Neoplastic; Contact Inhibition; Culture Media; Dextrans; Polyomavirus; Simian virus 40

Using a series of cold-sensitive variants of chemically transformed BHK-21 cells, revertants to the normal phenotype derived from a dimethyl-nitrosamine transformed clone of BHK-21 as well as revertants to the normal phenotype derived from polyoma transformed BHK-21 cells we have demonstrated that the surface phenotype described by enhanced agglutinability with Con A and WGA can be dissociated from the transformed phenotype described by anchorage independence (growth in semisolid medium). Specifically we have demonstrated that the surface characteristic of enhanced agglutinability may be found in a variety of cell lines which fail to display to grow in agar. Our work clearly shows that the two phenotypes described are not concomitantly controlled and tends to suggest that the phenotype of enhanced lectin agglutinability may be dissociated from the transformed phenotype.

Topics: Agar; Agglutination; Animals; Cell Division; Cell Line; Cell Transformation, Neoplastic; Clone Cells; Cold Temperature; Concanavalin A; Cricetinae; Lectins; Nitrosamines; Phenotype; Plant Lectins; Polyomavirus; Triticum

Topics: Agar; Animals; Cell Aggregation; Cell Line; Cell Transformation, Neoplastic; Cricetinae; Culture Media; DNA Viruses; Humans; Methylcholanthrene; Mice; Oncogenic Viruses; Rats; RNA Viruses; Sarcoma Viruses, Murine

We have tested the effects of exogenous proteases on the growth of normal and transformed hamster fibroblasts in the classic culture assays for transformation. The results indicate that exogenous proteases act to decrease the serum requirement of normal cells but not nearly to the extent that occurs in the process of viral transformation. Proteases do not further decrease the serum requirement of transformed cells, nor do they affect the maximal saturation density or the plating efficiency in soft agar of either normal or transformed cells. Under conditions optimal for growth stimulation, proteases decrease the strength of cell-to-substrate adhesion but do not affect cellular morphology. In contrast to previous studies, experiments using highly purified trypsin and several different active-site inhibitors strongly suggest that the growth-stimulatory activity of trypsin is not directly related to the proteolytic activity of the molecule.

Topics: Agar; Cell Adhesion; Cell Division; Cell Line; Cell Transformation, Neoplastic; Contact Inhibition; Culture Media; Fibroblasts; Pepsin A; Peptide Hydrolases; Pronase; Trypsin

Topics: Adenine; Adenine Phosphoribosyltransferase; Agar; Animals; Cell Adhesion; Cell Line; Cell Transformation, Neoplastic; Cells, Cultured; Cricetinae; Culture Media; Genes; Hypoxanthine Phosphoribosyltransferase; Mesocricetus; Mutation

Topics: Agar; Animals; Cell Adhesion; Cell Line; Cell Transformation, Neoplastic; Chromosomes; Cricetinae; Metaphase; Phenotype; Transformation, Genetic

Three parameters were evaluated as diagnostic of the malignant potential of cultured rat liver epithelial cells: cytology, growth in soft agar, and production of extracellular plasminogen activator. A total of 22 tumorigenic and nontumorigenic cultures from 15 cell lines were sent coded from their originators to two different laboratories for the evaluation of these three parameters. Cytologic diagnosis and growth in soft agar were reliable means of determining the malignant potential of the cultured cells. However, the production of extracellular plasminogen activator showed little correlation with tumorigenicity. Of cytologic properties evaluated, the two that correlated best with malignant potential were increased cytoplasmic basophilia and and increased nuclear:cytoplasmic ratio.

Topics: Agar; Animals; Cell Division; Cell Line; Cell Transformation, Neoplastic; Epithelium; Liver Neoplasms; Neoplasms, Experimental; Plasminogen Activators; Rats

We demonstrated that the productive infection of three different mammalian cell lines with two separate leukemia viruses is sufficient to induce a change in surface architecture that may be detected as enhanced agglutinability with two different plant lectins. Subsequent transformation of one of these cell lines with a chemical carcinogen did not further modify the agglutinability of the cell lines. Using a polyoma virus-transformed derivative of one of the parental lines, we have demonstrated that the LETS protein (whose absence from the surface membrane has been considered a marker of the transformed phenotype) may be present in cells displaying the capacity to plate in soft agar.

Topics: Agar; Agglutination; Animals; Carcinogens; Cell Division; Cell Line; Cell Membrane; Cell Transformation, Neoplastic; Cells, Cultured; Concanavalin A; Glycoproteins; Iodine Radioisotopes; Lactoperoxidase; Lectins; Leukemia Virus, Murine; Molecular Weight; Neoplasm Proteins; Nitroquinolines; Rodentia; Surface Properties

Various potential in vitro correlates of malignancy were studied in four chemically transformed C3H/10T1/2 Clone 8 mouse cell lines and were compared with controls cells. The degree of tumorigenicity was best predicted by the relative plating efficiencies of the morphologically transformed cells in soft agar. All transformed cells also showed an increase in extracellular fibrinolytic activity which may be an additional marker for transformation. Intracellular fibrinolytic activity and loss of 125I-labeled cell surface protein (M.W. 250,000) were not correlated with morphological transformation or tumorigenicity in these cells.

Topics: Agar; Cell Division; Cell Membrane; Cell Transformation, Neoplastic; Clone Cells; Fibrinolysis; Glycoproteins; Molecular Weight; Neoplasm Proteins; Neoplasms, Experimental

Cells cultured from various human and nonhuman malignant and normal tissues as well as mammalian cells transformed in vitro were examined for their ability to induce fibrinolysis. Generally, except for normal cells derived from lung or kidney, malignant cells had a greater ability to induce fibrinolysis than did their normal counterparts. A correlation existed between the abilities of the cells to induce fibrinolysis, grow in soft agar, and form tumors in immunosuppressed hosts.

Topics: Agar; Animals; Cell Transformation, Neoplastic; Cells, Cultured; Cricetinae; Dogs; Fibrinolysin; Fibrinolysis; Haplorhini; Humans; Kidney; Lung; Mice; Neoplasms, Experimental; Plasminogen

Epithelial cultures established from adult rat liver and from rat hepatomas induced in vivo with aromatic amine carcinogens have been compared by light and electron microscopy and by growth properties in liquid medium and in agar. The morphology and growth patterns of all of these cultures indicate that they have characteristics of epithelial rather than fibroblast cells. The criteria generally used to score for transformation of fibroblasts were not satisfactory for distinguishing normal epithelial cells from hepatoma cells in culture. Growth in agar, however, provides a simple and objective method of scoring for transformed epithelial cells, because only the tumorigenic cells grow in agar. Since none of the normal cultures had hydrocortisone-inducible tryosine aminotransferase, we lack definitive evidence that they are derived from liver parenchymal cells. The outstanding feature in the ultrastructure of the hepatoma cells in culture was the presence of type A and C viral particles. Whereas five hepatoma cultures and a spontaneously transformed normal liver cell line were positive for these particles, five independently isolated cell cultures from normal adult rat liver were negative. Evidence is presented that the viral particles seen in hepatoma cultures are due to activation of latent viruses rather than to in vitro contamination. The possible significance of these particles in hepatocarcinogenesis is discussed.

Topics: Agar; Animals; Carcinogens; Carcinoma, Hepatocellular; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Epithelial Cells; Epithelium; Liver; Liver Neoplasms; Microscopy, Electron; Neoplasms, Experimental; Oncogenic Viruses; Rats; Retroviridae

The TE-85 osteosarcoma cell line has several of the in vitro properties of malignant cells, including colony formation in agar, but has low extracellular fibrinolytic activity and no capacity to form tumors in ATS-treated hamsters. Some TE-85 cells clones (clones 2, 4 and 6) have increased fibrinolytic activity but do not form tumors in hamsters. TE-85 cells infected with mammalian transformation-defective viruses have low (FeLV) or increased (RD-114 virus) levels of fibrinolytic activity and do not form tumors in hamsters. TE-85 cell either nonproductively infected with Ki-MSV or productively infected with M-MSV (RD-114), have fibrinolytic activity and can form tumors in hamsters. The MSV gene(s) but not colony formation in agar or extracellular fibrinolytic activity appears to be capable of rendering TE-85 cells tumorigenic in ATS-treated hamsters.

Topics: Agar; Animals; Antilymphocyte Serum; Cell Line; Cell Transformation, Neoplastic; Clone Cells; Cricetinae; Culture Media; Fibrinolysis; Humans; Immunosuppression Therapy; In Vitro Techniques; Neoplasm Transplantation; Neoplasms, Experimental; Oncogenic Viruses; Osteosarcoma; T-Lymphocytes; Transplantation, Heterologous

A clone of spontaneously transformed Chinese hamster lung cells was exposed to N-methyl-N'-nitro-N-nitroso-guanidine (MNNG), and six heat-sensitive and three cold-sensitive mutants were isolated after selection for inability to form colonies in soft agar at 39.5 degrees C and 34.5 degrees C, respectively. The heat-sensitive mutants had growth characteristics of transformed cells at 34.5 degrees C, but exhibited a normal phenotype at 39.5 degrees C. By contrast, cold-sensitive mutants displayed the characteristics of the normal cells at 34.5 degrees C and converted to a transformed phenotype at 39.5 degrees C. Transformed parent cells exhibited no obvious temperature-dependent properties. Temperature shift experiments showed that the colony-forming ability of both types of mutants was fully reversible. All of the mutants were able to grow well at both permissive and nonpermissive temperatures when grown on the surface of plastic dishes. Such mutants will be useful in analysis of factors involved in the expression of the transformed state or the maintenance of the nontransformed state.

Topics: Agar; Animals; Bromodeoxyuridine; Cell Division; Cell Line; Cell Transformation, Neoplastic; Clone Cells; Cold Temperature; Cricetinae; Fibroblasts; Hot Temperature; Lung; Methylnitronitrosoguanidine; Mutagens; Mutation; Phenotype

Topics: Agar; Cell Count; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Chromosomes; Clone Cells; Culture Media; Karyotyping

Topics: Agar; Bone Marrow Cells; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Clone Cells; Humans; In Vitro Techniques; Leukemia, Monocytic, Acute; Leukemia, Myeloid; Leukemia, Myeloid, Acute; Leukocytes

Topics: Agar; Agglutination Tests; Animals; Bromodeoxyuridine; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cells, Cultured; Chromatography, Paper; Concanavalin A; Mice; Neoplasms, Experimental; Polyomavirus

Topics: Agar; Alpharetrovirus; Animals; Avian Leukosis Virus; Avian Sarcoma Viruses; Cell Transformation, Neoplastic; Chick Embryo; Culture Techniques; Defective Viruses; Mutation; Temperature; Viral Plaque Assay

Topics: Agar; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Cyclic AMP; Humans; Rhabdomyosarcoma

Topics: Agar; Alpharetrovirus; Animals; Avian Leukosis Virus; Avian Sarcoma Viruses; Bone Marrow; Cell Transformation, Neoplastic; Cells, Cultured; Chick Embryo; Chickens; Clone Cells; Female; Hematopoietic System; Microscopy, Phase-Contrast; Neutralization Tests; Satellite Viruses; Spleen; Viral Plaque Assay; Vitelline Membrane

Topics: Adsorption; Agar; Animals; Cell Line; Cell Transformation, Neoplastic; Cricetinae; Kidney; Mutation; Polyomavirus; Temperature; Time Factors; Virus Replication

Fetal bovine and dog serum were selectively freed of plasminogen by affinity chromatography. The resulting serum as well as native and reconstituted serum (obtained by the addition of purified plasminogen to the plasminogen-depleted serum) were used to examine the role of plasminogen in (a) growth of normal and SV-40-transformed hamster embryo fibroblasts in liquid medium, (b) growth of SV-40-transformed hamster embryo fibroblasts in soft agar, (c) aggregation - a characteristic morphological change of SV-40-transformed hamster cells, and (d) migration of SV-40-transformed and control 3T3 cells from a monolayer into a "wound." The results demonstrated that exponential growth of both normal and transformed cells in liquid medium proceeded at the same rate in the presence or absence of plasminogen. In contrast, removal of plasminogen markedly depressed the plating efficiency of transformed cells in soft agar, eliminated their characteristic aggregation, and substantially reduced the extent of migration. The role of plasminogen and its activation in oncogenic transformation is discussed.

Topics: Agar; Animals; Cattle; Cell Aggregation; Cell Division; Cell Movement; Cell Transformation, Neoplastic; Cells, Cultured; Chromatography, Affinity; Cricetinae; Dogs; Embryo, Mammalian; Fetus; Fibrinolysis; Fibroblasts; Iodine Radioisotopes; Plasminogen; Simian virus 40

Topics: Agar; Agglutination Tests; Animals; Ascitic Fluid; Cell Transformation, Neoplastic; Cells, Cultured; Chickens; Concanavalin A; DNA; Fluorescent Antibody Technique; Genetic Variation; Hot Temperature; Interferons; Macrophages; Mice; Mice, Inbred C57BL; Mice, Inbred Strains; Polyomavirus; Thymidine; Time Factors; Tritium

Topics: Agar; Alkanesulfonates; Animals; Cell Count; Cell Line; Cell Transformation, Neoplastic; Cells, Cultured; Colchicine; Cricetinae; Cytological Techniques; Kidney; Mitosis; Mutagens; Mutation; Polyomavirus; Temperature

Topics: Agar; Animals; Avian Sarcoma Viruses; Cell Line; Cell Transformation, Neoplastic; Clone Cells; Cricetinae; Cytological Techniques; Female; Fibroblasts; HeLa Cells; Humans; Kidney; L Cells; Leukemia, Lymphoid; Lung; Mammary Neoplasms, Experimental; Methods; Mice; Mice, Inbred C3H; Mice, Inbred DBA; Ovary; Polyomavirus; Rats; Sarcoma, Yoshida

Cells of the methylene dimethanesulphonate-(MDMS)-resistant Yoshida sarcoma cell line contain a low molecular weight "resistance factor" which is present in the culture medium of these cells and may be utilized by MDMS-sensitive Yoshida sarcoma cells either by co-culturing the two cell lines or by culturing the MDMS-sensitive Yoshida cells in a medium containing 20% used medium of MDMS-resistant Yoshida cells or in the presence of dialysed medium from resistant cells. The "resistance factor" does not inactivate the drug itself or its metabolites, and it has no influence on the sensitivity of the cells if added after MDMS treatment. Twenty-four hours seems to be enough time for the transfer of the resistance factor, but its effect on whole populations decreases within 24 hours of ceasing the supply. The relationship between these findings and the known phenomena of metabolic co-operation are discussed.

Topics: Agar; Animals; Cell Line; Cell Transformation, Neoplastic; Culture Techniques; Drug Resistance; Esters; Immunity, Cellular; Sarcoma, Yoshida; Sulfonic Acids; Time Factors; Tritium

Topics: Agar; Animals; Antigens; Antigens, Viral; Cell Line; Cell Transformation, Neoplastic; Centrifugation, Density Gradient; Complement Fixation Tests; Erythrocytes; Gammaretrovirus; Helper Viruses; Hemagglutination Tests; Immune Sera; Immunodiffusion; Leukemia Virus, Murine; Mice; Rabbits; Sheep; Sucrose

Topics: Agar; Animals; Cat Diseases; Cats; Cell Line; Cell Transformation, Neoplastic; Cells, Cultured; Clone Cells; Cytopathogenic Effect, Viral; Gammaretrovirus; Helper Viruses; Kidney; Oncogenic Viruses; RNA Viruses; Viral Interference

Topics: Agar; Animals; Cell Transformation, Neoplastic; Cells, Cultured; Chromosomes; Clone Cells; Cricetinae; Hot Temperature; Polyomavirus

Chick embryo cells infected with a mutant (Ta) of the Bryan high-titer strain of Rous sarcoma virus (RSV-BH) are morphologically transformed at 36 C but appear similar to uninfected cells at 41 C. When cells infected with RSV-BH-Ta are switched from 41 to 36 C, morphological changes characteristic of transformation are observable within 10 min. The transformation is reversible; cells shifted from 36 to 41 C have been observed to lose their transformed morphology within 1 hr. The transformation after a shift in temperature is unaffected by inhibition of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or protein synthesis, demonstrating that the proteins involved in the morphological change are already present. Transformed cells infected with RSV-BH or RSV-BH-Ta take up hexose and synthesize hyaluronic acid at higher rates than uninfected cells or RSV-BH-Ta-infected cells grown at 41 C. However, inhibition of either protein or RNA synthesis, but not DNA synthesis, prevented the induction of increased hexose uptake and hyaluronic acid synthesis after a shift of RSV-BH-Ta-infected cells from 41 to 36 C. Therefore, these biochemical changes are secondary to a more basic change responsible for morphological transformation.

Topics: Agar; Animals; Avian Sarcoma Viruses; Cell Transformation, Neoplastic; Cells, Cultured; Chick Embryo; Cycloheximide; Cytarabine; Dactinomycin; DNA; Genetics, Microbial; Glucose; Hyaluronic Acid; Microscopy, Phase-Contrast; Mutation; Protein Biosynthesis; Puromycin; RNA; Temperature; Time Factors; Tritium; Virus Replication

Topics: Agar; Animals; Blood; Bone Marrow; Bone Marrow Cells; Cattle; Cell Division; Cell Line; Cell Transformation, Neoplastic; Clone Cells; Cricetinae; Dose-Response Relationship, Drug; Erythrocytes; Horses; Male; Mice; Mice, Inbred C57BL; Polyomavirus; Rabbits; Rats; Sheep; Swine

Topics: Agar; Animals; Antigens, Neoplasm; Cell Line; Cell Transformation, Neoplastic; Chromosomes; Cricetinae; Culture Media; Dactinomycin; Drug Resistance; Fluorescent Antibody Technique; Graft Rejection; Histocompatibility Antigens; Karyotyping; Simian virus 40; Time Factors

A cell line, derived from a spontaneous equine connective tissue tumor (equine sarcoid), has been established. The morphological and growth characteristics indicative of malignant transformation of the cells include a disoriented, rapid growth and loss of contact inhibition. Further evidence of transformation is the agglutination of these cells by concanavalin A and their ability to divide in semisolid media.

Topics: Agar; Agglutination Tests; Animals; Cell Line; Cell Transformation, Neoplastic; Concanavalin A; Culture Media; Culture Techniques; Horses; Male; Skin Neoplasms; Trypsin

Topics: Agar; Animals; Benz(a)Anthracenes; Benzopyrans; Cell Count; Cell Transformation, Neoplastic; Chromosomes; Clone Cells; Cricetinae; Culture Media; Culture Techniques; Female; Fibrosarcoma; Karyotyping; Male; Methods; Mitosis; Neoplasm Transplantation; Neoplasms, Experimental; Polycyclic Compounds; Transplantation, Homologous

Topics: Agar; Animals; Bromodeoxyuridine; Cell Division; Cell Line; Cell Survival; Cell Transformation, Neoplastic; Clone Cells; Cricetinae; Culture Media; Culture Techniques; DNA; Kidney; Light; Methods; Methylcellulose; Polyomavirus; Trypsin

Topics: Agar; Animals; Antigens; Bromodeoxyuridine; Cell Division; Cell Line; Cell Nucleus; Cell Survival; Cell Transformation, Neoplastic; Chromosomes; Clone Cells; Complement Fixation Tests; Cricetinae; Culture Media; Culture Techniques; Genetic Variation; Kidney; Light; Methylcellulose; Mitosis; Oncogenic Viruses; Phenotype; Polyomavirus; Time Factors

Topics: Agar; Animals; Avian Sarcoma Viruses; Cell Transformation, Neoplastic; Cells, Cultured; Chick Embryo; Chickens; Culture Media; Cycloheximide; Fluorouracil; Genetics, Microbial; Hexoses; Mutagens; Mutation; Puromycin; Sarcoma, Avian; Temperature; Tritium; Virus Replication

Topics: Agar; Cell Transformation, Neoplastic; Centrifugation, Density Gradient; Clone Cells; Gammaretrovirus; Helper Viruses; Kidney; Moloney murine leukemia virus; RNA, Viral; Sucrose; Tritium; Uridine; Viral Interference; Virus Replication

Topics: Agar; Animals; Cell Line; Cell Transformation, Neoplastic; Culture Techniques; Gammaretrovirus; Helper Viruses; Kinetics; Methods; Mice; Moloney murine leukemia virus; Rats; Sarcoma, Experimental; Virus Cultivation

Topics: Agar; Animals; Avian Sarcoma Viruses; Cell Transformation, Neoplastic; Chick Embryo; Culture Media; Culture Techniques; Dextrans; Embryo, Mammalian; Embryo, Nonmammalian; Polysaccharides; Sulfates; Tissue Extracts

Topics: Agar; Animals; Cell Transformation, Neoplastic; Culture Techniques; Embryo, Mammalian; Leukemia Virus, Murine; Leukemia, Experimental; Mice; Microscopy, Electron; Neoplasm Transplantation; Thymus Gland

In cells transformed by ts-3, a thermosensitive mutant of polyoma virus, the loss of inhibition of DNA synthesis by topographical factors (topo-inhibition), is rendered temperature-dependent, providing evidence that the viral genome controls this essential aspect of transformation. The expression of two other attributes of transformed cells, growth in agar and serum requirement for initiation of DNA synthesis in a wound in culture, is not made temperature-dependent. In productive infection of BALB-3T3 cells by ts-3, virus-induced stimulation of cellular DNA synthesis and movement is rendered temperature-dependent.

Topics: Agar; Animals; Autoradiography; Blood; Cell Line; Cell Movement; Cell Transformation, Neoplastic; Cricetinae; Culture Media; Culture Techniques; DNA, Viral; Genetics, Microbial; Kidney; Mutation; Polyomavirus; Temperature; Thymidine; Tritium

Topics: Agar; Animals; Antineoplastic Agents; Asparaginase; Avian Sarcoma Viruses; Cattle; Cell Line; Cell Transformation, Neoplastic; Chick Embryo; Culture Media; Culture Techniques; Depression, Chemical; Immune Sera; Liver Extracts; Methotrexate; Mollusca; Phosphates; Time Factors

Topics: Agar; Animals; Antigens; Cell Line; Cell Transformation, Neoplastic; Clone Cells; Complement Fixation Tests; Culture Techniques; Fluorescent Antibody Technique; Liver; Lung; Mice; Neoplasm Transplantation; Papillomaviridae; Polyomaviridae

Topics: Agar; Animals; Cell Transformation, Neoplastic; Culture Techniques; Fibroblasts; Interferons; Mice; Microscopy, Electron; Moloney murine leukemia virus; Newcastle disease virus; Polysaccharides

Topics: Agar; Animals; Cell Differentiation; Cell Transformation, Neoplastic; Clone Cells; Culture Techniques; Feedback; Hematopoietic System; Leukemia; Mice; Neoplasms, Experimental

Topics: Agar; Animals; Buffers; Cell Transformation, Neoplastic; Clone Cells; Contact Inhibition; Cricetinae; Culture Media; DNA; Genetics, Microbial; Mutation; Phosphates; Polyomavirus; Radiation Effects; RNA