epidermal-growth-factor and Cell-Transformation--Viral

Topics: Amino Acid Sequence; Animals; Cell Division; Cell Transformation, Viral; Epidermal Growth Factor; Genes, Viral; Humans; Molecular Sequence Data; Skin

Cancer Research has changed substantially over the past several years since oncogenes were isolated from cancer cells. More than 40 oncogenes have been identified to date from tumor viruses and cancer cells. Many of the gene products seem related to signalling pathways that determine growth and differentiation of cells. This review attempts to summarize much of the currently available data and to gain future perspectives.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinogens; Cell Transformation, Neoplastic; Cell Transformation, Viral; Epidermal Growth Factor; Genes, Viral; Humans; Mammary Neoplasms, Experimental; Neoplasms; Platelet-Derived Growth Factor; Retroviridae; Transfection; Urinary Bladder Neoplasms

For cell growth and division to occur, a large variety of metabolic processes must be carefully coordinated in the cell. Through evolutionary pressures, specific hormones and growth factors have acquired the ability to trigger a complex coordinated "pleiotropic growth response" in their target cells. This complex response is mediated by specific cellular receptors and intracellular messengers. Teleologically then, it makes sense that in oncogenesis this growth regulating network is utilized by the production of proteins which mimic growth factors, the activated form of their receptors or, the messengers themselves. Several lines of evidence indicate that the epidermal growth factor-stimulated growth regulatory system is involved in cellular proliferation, both normal and neoplastic. Some of the effects of epidermal growth factor in carcinogenesis are separable from its direct, growth stimulatory effects. Thus, the role of epidermal growth factor in carcinogenesis is more complex than is its role in stimulating growth.

Topics: Animals; Carcinogens; Cell Transformation, Neoplastic; Cell Transformation, Viral; Epidermal Growth Factor; ErbB Receptors; Humans; Immune Tolerance; Neoplasms; Oncogene Proteins, Viral; Oncogenes; Peptides; Receptors, Cell Surface; Transforming Growth Factors

Different aspects of the interaction of polypeptide growth factors with their receptors are reviewed. The problem of structural and functional interactions of the normal and transforming growth factors as well as the protein products of oncogenes during transmittance of a mitogenic signal and the proliferation regulation of normal and tumour cells is discussed.

Topics: Animals; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cells, Cultured; DNA, Neoplasm; DNA, Viral; Drug Interactions; Epidermal Growth Factor; Gene Expression Regulation; Genes, Viral; Growth Substances; Male; Mice; Oncogenes; Oncogenic Viruses; Peptides; Platelet-Derived Growth Factor; Protein Biosynthesis; Receptors, Cell Surface; Transforming Growth Factors; Translocation, Genetic

Topics: Animals; Cell Division; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cells, Cultured; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation; Growth Substances; Hormones; Humans; Lymphocyte Activation; Mitogens; Oncogenes; Oncogenic Viruses; Receptors, Cell Surface

Cellular oncogenes and their activated and retrovirus-coded counterparts play an important role in cellular regulation. Here the relationship between such oncogenes and the genes coding for the transforming proteins of the papovaviruses, polyoma viruses, and simian virus 40 (SV40) is discussed. It is concluded that polyoma virus may transform established cells by a mechanism involving activation of a cellular oncogene product, whereas SV40 may transform by a mechanism involving a previously little studied cytoplasmic form of the transforming protein.

Topics: Animals; Antigens, Polyomavirus Transforming; Antigens, Viral, Tumor; Base Sequence; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; DNA; DNA-Binding Proteins; DNA, Viral; Epidermal Growth Factor; Humans; Neoplasms; Oncogenes; Papillomaviridae; Platelet-Derived Growth Factor; Polyomaviridae; Polyomavirus; Protein Kinases; Protein-Tyrosine Kinases; Simian virus 40; Transcription, Genetic; Viral Proteins

Topics: Adenosine Triphosphate; Animals; Avian Sarcoma Viruses; Cations, Divalent; Cell Membrane; Cell Transformation, Viral; Cyclic AMP; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Insulin; Macromolecular Substances; Molecular Weight; Phosphoprotein Phosphatases; Phosphorylation; Protein Kinases; Protein-Tyrosine Kinases; Receptor, Insulin; Receptors, Cell Surface; Serine; Substrate Specificity; Tyrosine

PDGF is a potent mitogen that initiates the proliferation of quiescent fibroblastic cells. EGF and somatomedin C (or insulin) can replace the requirement for plasma to function synergistically with PDGF to stimulate DNA synthesis. PDGF, EGF and somatomedin C control discrete cellular events in the cell cycle. Cyclic AMP can potentiate the effects of polypeptide mitogens. The down-regulation of EGF receptors by PDGF and cyclic AMP brings about a loss of the requirement for exogenous EGF. The transient treatment of density-arrested fibroblasts with PDGF allows better study of synergistic actions of PDGF and plasma-derived factors. These synergistic interactions are important to understand in determining how multiple growth factors regulate cellular proliferation.

Topics: Animals; Blood; Calcium; Cell Cycle; Cell Division; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cells, Cultured; Cyclic AMP; DNA; Drug Synergism; Epidermal Growth Factor; ErbB Receptors; Fibroblast Growth Factors; Fibroblasts; Growth Substances; Humans; Insulin-Like Growth Factor I; Mice; Models, Biological; Platelet-Derived Growth Factor; Protein Biosynthesis; Receptors, Cell Surface; Receptors, Platelet-Derived Growth Factor; Somatomedins

Topics: Animals; Carcinogens; Cell Adhesion; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cells, Cultured; Cocarcinogenesis; DNA, Neoplasm; Epidermal Growth Factor; Humans; Methylation; Phenotype

EGF-Rs are cell membrane glycoproteins of wide distribution. They have not yet been fully characterized or purified but are probably molecules of 170-190,000 mol. wt. in most cells. The growth factor EGF binds and will saturate cell surface receptors with a KA of about 5 X 10(9) M-1 although a receptor class with an affinity in excess of 10(10) M-1 has been detected in some cells. The number of receptors on a cell does not determine the level of its response. Some cell types have receptors which bind EGF, but with no mitogenic response. The ways in which receptor affinity and/or number is modulated are described. This and other evidence is reviewed in a search for a suitable model of a mechanism of action on the cell, which best fits the current data. There is ample evidence that EGF binds to the receptor; that ligand-receptor complexes cluster or aggregate; and then are internalized and degraded, but evidence for a direct connection between internalization and the subsequent mitogenic response is lacking. Good correlations between internalization and mitogenic responses have been observed and developed into a theory of endocytic activation, but there is a body of evidence which cannot be accommodated by this theory. Instead, an alternative model is suggested.

Topics: Animals; Binding Sites; Cell Membrane; Cell Transformation, Neoplastic; Cell Transformation, Viral; DNA; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Kinetics; Models, Biological; Molecular Weight; Organ Specificity; Peptides; Pregnancy; Receptors, Cell Surface; Species Specificity; Teratoma; Tissue Distribution; Tretinoin

Topics: Adipose Tissue; Animals; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cyclic AMP; Epidermal Growth Factor; ErbB Receptors; Insulin; Membrane Proteins; Models, Biological; Oncogene Protein pp60(v-src); Phosphorylation; Protein Kinases; Proteins; Receptor, Insulin; Receptors, Cell Surface; Sodium-Potassium-Exchanging ATPase; Viral Proteins

Cell-to-cell contact can result in a variety of changes in the cell's physiology. For different cell types, this may include both the initiation as well as the cessation of cell growth and changes in the state of differentiation. This review examines in detail one such phenomenon, density-dependent inhibition of growth, which is observed with many fibroblasts in culture. Data are summarized which demonstrate that the cessation of growth at high cell density is in part a consequence of cell-to-cell contact. An approach to the study of the molecular basis of this phenomenon is presented based on the demonstration that plasma membranes, when bound to sparse growing cells, mimic contact inhibition of growth. The present status of attempts to purify plasma membrane proteins responsible for this effect are summarized, and the properties of these membrane proteins are compared to those of previously described "soluble" proteins that inhibit cellular growth.

Topics: Animals; Cell Communication; Cell Division; Cell Membrane; Cell Transformation, Viral; Cells, Cultured; Contact Inhibition; Epidermal Growth Factor; Growth Substances; Humans; Kinetics; Membrane Proteins; Mice; Mitogens; Peptides; Platelet-Derived Growth Factor; Simian virus 40

The molecular scaffold kinase suppressor of Ras 1 (KSR1) regulates the activation of the Raf/MEK/extracellular signal-regulated kinase (ERK) signal transduction pathway. KSR1 disruption in mouse embryo fibroblasts (MEFs) abrogates growth factor-induced ERK activation, H-Ras(V12)-induced replicative senescence, and H-Ras(V12)-induced transformation. Caveolin-1 has been primarily described as a major component of the coating structure of caveolae, which can serve as a lipid binding adaptor protein and coordinates the assembly of Ras, Raf, MEK, and ERK. In this study, we show that KSR1 interacts with caveolin-1 and is responsible for MEK and ERK redistribution to caveolin-1-rich fractions. The interaction between KSR1 and caveolin-1 is essential for optimal activation of ERK as a KSR1 mutant unable to interact with caveolin-1 does not efficiently mediate growth factor-induced ERK activation at the early stages of pathway activation. Furthermore, abolishing the KSR1-caveolin-1 interaction increases growth factor demands to promote H-Ras(V12)-induced proliferation and has adverse effects on H-Ras(V12)-induced cellular senescence and transformation. These data show that caveolin-1 is necessary for optimal KSR1-dependent ERK activation by growth factors and oncogenic Ras.

Topics: Animals; Caveolae; Caveolin 1; Cell Transformation, Viral; Cells, Cultured; Cellular Senescence; Embryo, Mammalian; Epidermal Growth Factor; Gene Expression Regulation; HEK293 Cells; Humans; MAP Kinase Signaling System; Mice; Protein Kinases; ras Proteins

Ameloblastoma is an odontogenic tumor characterized by local invasiveness and frequent recurrence. The surrounding stroma, composed of different cell types and extracellular matrix (ECM), may influence ameloblastoma invasive behavior. Furthermore, tumor and stromal cells secrete matrix metalloproteases (MMPs), which, in turn, can modulate the matrix and promote the release of ECM-bound growth factors. Among these growth factors, epidermal growth factor (EGF) and its receptor, EGFR, have already been shown to stimulate MMP synthesis, suggesting that an interdependent mechanism, involving MMP activity and growth factors release, may contribute to tumor invasiveness. The aim of this study was to evaluate the effects of the EGF/EGFR signaling pathway on migration, invasion, and MMP activity, in a primary cell line derived from human ameloblastoma. We established and characterized a primary cell line (AME-1) from a human ameloblastoma sample. This cell line was transduced with human papillomavirus type 16 (HPV16) E6/E7 oncogenes, generating the AME-HPV continuous cell line. EGF, MMP2, and MMP9 expression in ameloblastoma biopsies and in the AME-HPV cell line was analyzed by immunohistochemistry and immunofluorescence, respectively. Migratory activity of EGF-treated AME-HPV cells was investigated using monolayer wound assays and Transwell chambers. EGF-induced invasion was assessed in Boyden chambers coated with Matrigel. Conditioned medium from EGF-treated cells was subjected to zymography. EGFR expression in AME-HPV cells was silenced by small interfering RNA (siRNA), to verify the relationship between this receptor and MMP secretion. Ameloblastoma samples and AME-HPV cells expressed EGF, EGFR, MMP2, and MMP9. AME-HPV cells treated with EGF showed increased rates of migration and invasion, as well as enhanced MMP2 and MMP9 activity. EGFR knockdown decreased MMP2 and MMP9 levels in AME-HPV cells. EGFR signaling downstream of EGF probably regulates migration, invasion, and MMP secretion of ameloblastoma-derived cells.

Topics: Ameloblastoma; Blotting, Western; Cell Movement; Cell Proliferation; Cell Transformation, Viral; Epidermal Growth Factor; ErbB Receptors; Fluorescent Antibody Technique; Humans; Jaw Neoplasms; Matrix Metalloproteinases; Neoplasm Invasiveness; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Tumor Cells, Cultured; Wound Healing

Normal human epithelial cell cultures exhibit a limited (although different between tissues) lifespan in vitro. In previous studies, urothelial cell cultures were immortalized using retroviral transformation with human papillomavirus type 16 E6 and E7 genes, in undefined culture systems containing serum or bovine pituitary extract.. Due to the variability of results in such systems, we instead developed a procedure for the immortalization of urothelial cells using a defined, serum-free culture system.. Immortalization through retroviral transformation with human papillomavirus type 16 E6 and E7 was successful, and transformation of urothelial cells conferred an extended over normal lifespan and restored telomerase activity. Transformed cells retained typical morphology and exhibited a similar growth rate, cytokeratin immunoreactivity pattern, and response to growth factors as observed in untransformed cells. Karyotype analysis revealed a gradual accumulation of genetic mutations that are consistent with previously reported mutations in epithelial cells transformed with human papillomavirus type 16 E6 and E7.. The ability to extend the in vitro lifespan of cells holds the potential to reduce the continuous need for tissue samples and to enable complete investigations with one cell line.

Topics: Animals; Cell Cycle; Cell Growth Processes; Cell Line, Transformed; Cell Transformation, Viral; Cells, Cultured; Clone Cells; Culture Media, Serum-Free; Epidermal Growth Factor; ErbB Receptors; Genes, Viral; Human papillomavirus 16; Humans; Karyotyping; Keratins; Mice; Oncogene Proteins, Viral; Papillomavirus E7 Proteins; Papillomavirus Infections; Phenotype; Quinazolines; Repressor Proteins; Telomerase; Urothelium

Infection of keratinocytes with high risk human Papilloma virus causes immortalization, and when followed by further mutations, leads to cervical cancer and other anogenital tumors. Here we monitor the progressive loss of robustness in an in vitro model of the early stages of transformation that comprises normal keratinocytes and progressive passages of HPV16 immortalized cells. As transformation progresses, the cells acquire higher proliferation rates and gain the ability to grow in soft agar. Concurrently, the cells lose robustness, becoming more sensitive to serum starvation and DNA damage by Cisplatin. Loss of robustness in the course of transformation correlates with significant reductions in the activities of the anti-apoptotic proteins PKB/Akt, Erk, Jnk and p38 both under normal growth conditions and upon stress. In parallel, loss of robustness is manifested by the shrinkage of the number of growth factors that can rescue starving cells from apoptosis, with the emergence of dependence solely on IGF1. Treatment with IGF1 activates PKB/Akt and Jnk and through them inhibits p53, rescuing the cells from starvation. We conclude that transformation in this model induces higher susceptibility of cells to stress due to reduced anti-apoptotic signaling and hyper-activation of p53 upon stress.

Topics: Apoptosis; Cell Cycle; Cell Division; Cell Transformation, Viral; Epidermal Growth Factor; Genes, Reporter; Genome, Viral; Human papillomavirus 16; Humans; Immunoblotting; Insulin-Like Growth Factor I; Keratinocytes; Kinetics; MAP Kinase Kinase 4; p38 Mitogen-Activated Protein Kinases; Polymerase Chain Reaction; Proto-Oncogene Proteins c-akt; Transfection

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is known to cause a large number of adverse effects, mediated largely by its binding to the aryl-hydrocarbon receptor (AhR) and subsequent modulation of gene expression. It is thought that AhR mediates these effects through the untimely and disproportionate expression of specific genes. However, the exact mechanism, or the genes involved, through which TCDD leads to these effects is still unknown. This study reports the discovery of a novel target gene, epiregulin, which is regulated by TCDD-activated AhR. Epiregulin is a growth regulator which belongs to the epidermal growth factor (EGF) family. Using real time quantitative PCR (qPCR), it was established that TCDD upregulates epiregulin gene expression. The promoter region of epiregulin has a dioxin responsive element (DRE) 56 nucleotides upstream of the transcription start site, along with three potential Sp1 binding sites. Chromatin immunoprecipitation (ChIP) assays with an anti-AhR antibody showed promoter occupancy upon TCDD treatment. Luciferase reporter assays using a vector harboring the first 125 base pairs of the epiregulin rat promoter revealed an increase in signal on TCDD treatment, which was lost upon mutation of the DRE. Epiregulin and TCDD treatment mediated a dose-dependent increase in primary mouse keratinocyte growth. These results demonstrate that AhR directly increases epiregulin expression, which could play an important role in TCDD mediated tumor promotion observed in rodent models.

Topics: Animals; Animals, Newborn; Cell Line, Transformed; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Viral; Chromatin Immunoprecipitation; Dose-Response Relationship, Drug; Epidermal Growth Factor; Epiregulin; Hepatocytes; Keratinocytes; Mice; Mice, Inbred C57BL; Mitogens; NIH 3T3 Cells; Polychlorinated Dibenzodioxins; Receptors, Aryl Hydrocarbon; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transcription, Genetic; Up-Regulation

The proteome of a proliferating human stem cell line was analyzed and then utilized to detect stem cell differentiation-associated changes in the protein profile. The analysis was conducted with a stable human fetal midbrain stem cell line (ReNcell VM) that displays the properties of a neural stem cell. Therefore, acquisition of proteomic data should be representative of cultured human neural stem cells (hNSCs) in general. Here we present a 2-DE protein-map of this cell line with annotations of 402 spots representing 318 unique proteins identified by MS. The subsequent proteome profiling of differentiating cells of this stem cell line at days 0, 4 and 7 of differentiation revealed changes in the expression of 49 identified spots that could be annotated to 45 distinct proteins. This differentiation-associated expression pattern was validated by Western blot analysis for transgelin-2, proliferating cell nuclear antigen, as well as peroxiredoxin 1 and 4. The group of regulated proteins also included NudC, ubiquilin-1, STRAP, stress-70 protein, creatine kinase B, glial fibrillary acidic protein and vimentin. Our results reflect the large rearrangement of the proteome during the differentiation process of the stem cells to terminally differentiated neurons and offer the possibility for further characterization of specific targets driving the stem cell differentiation.

Topics: Blotting, Western; Cell Differentiation; Cell Line; Cell Line, Transformed; Cell Proliferation; Cell Transformation, Viral; Computational Biology; Culture Media; Databases, Protein; Electrophoresis, Gel, Two-Dimensional; Epidermal Growth Factor; Fibroblast Growth Factor 2; Genetic Markers; Humans; Mass Spectrometry; Mesencephalon; Microfilament Proteins; Muscle Proteins; Neoplasm Proteins; Neurons; Peptide Mapping; Peroxidases; Peroxiredoxins; Proliferating Cell Nuclear Antigen; Proteome; Retroviridae; Selection, Genetic; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Stem Cells; Transduction, Genetic; Transgenes; Trypsin

The cell adhesion molecule T-cadherin is an unusual member of the cadherin superfamily that lacks a cytoplasmic domain, binding instead to the cell membrane via a glycophosphatidyl inositol anchor. T-cadherin is a receptor for hexameric Acrp30/adiponectin and binds low-density lipoproteins in endothelial cells. T-cadherin is expressed widely in the brain and cardiovascular system, but expression is absent or decreased in several cancers. Little is known about the mechanisms and factors that control T-cadherin expression. Therefore, to investigate regulation of T-cadherin expression, we analysed 3.9 kb of the 5'-flanking region of human T-cadherin for promoter activity and identified potential transcription factor binding sites. Western blotting and a quantitative real-time RT-PCR assay developed for T-cadherin showed that estradiol, progesterone, EGF, dexamethasone and factors in serum were involved in transcriptional and post-transcriptional regulation of T-cadherin in human osteosarcoma cells; the effects observed were opposite to those described for T-cadherin's ligand, adiponectin. The data suggest that T-cadherin is regulated in a complex manner indicative of a role in hormone and drug-induced changes in bone morphology and pathology.

Topics: 5' Flanking Region; Animals; Cadherins; Cell Adhesion; Cell Line, Transformed; Cell Line, Tumor; Cell Transformation, Viral; Chlorocebus aethiops; COS Cells; Dexamethasone; Epidermal Growth Factor; Estradiol; Gene Expression Regulation; Genes, Reporter; Glucocorticoids; Hormones; Humans; Luciferases; Osteosarcoma; Progesterone; Promoter Regions, Genetic; RNA, Messenger

To search for potential biomarkers used to monitor the process of immortalization, we investigated the relative level of telomerase activity and other immortal phenotypes in the SHEE esophageal epithelial cell line. This human fetal esophageal epithelial cell line, induced by human papilloma virus (HPV) 18 E6E7, was continually propagated over 100 passages. Fourteenth passage cells (SHEE14) were cultured in a flask with a serum-free medium and continually cultured to the 30th passage (SHEE30). Cells of SHEE14, SHEE20 and SHEE30 were examined according to cell morphology, cell cycle, apoptosis, contact-inhibition growth, anchorage- dependency, dose-dependency to epithelial growth factors (EGF), telomerase activity and tumorigenicity. The SHEE14 cells exhibited good differentiation with contact-inhibition and anchorage-dependent growth. The SHEE20 cells exhibited increase of senescent and apoptotic cells, and difficulty in propagation. The SHEE30 cells exhibited a higher proliferative index and some undifferentiated cells, with weakened contact-inhibition and anchorage-dependent growth. The telomerase was activated in cells of SHEE30, but not in SHEE14 and SHEE20 cells. The different response to dose-dependency to EGF was not statistically different in SHEE14 and SHEE30. Three groups of cells displayed lack of tumor formation in nude mice. Compared with SHEE14 and SHEE20, SHEE30 cells were of immortalized status with immortal phenotype, which consisted of telomerase activity, increase of cell proliferation, weakened contact-inhibition and anchorage-dependent growth, dose dependency to EGF and lack of tumor formation. From passage 14 to 30th passage, SHEE cells went through cellular senescence, apoptosis and immortalization. With a view toward diagnostic and biological aspects, telomerase activity is a crucial step and a cardinal requirement for immortalization. The telomerase activity and other immortal phenotypes are potential markers for monitoring the process of immortalization.

Topics: Animals; Biomarkers; Cell Cycle; Cell Division; Cell Line; Cell Transformation, Viral; Cell Transplantation; Cellular Senescence; Colony-Forming Units Assay; Contact Inhibition; Epidermal Growth Factor; Epithelial Cells; Esophagus; Fetus; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Papillomaviridae; Phenotype; Telomerase

To determine in SV40-immortalized rabbit corneal epithelial cells (RCE), whether there is conservation of parent tissue serum growth-factor-stimulated cytokine receptor activation and downstream intracellular signaling events mediating control of cell cycle progression and differentiation.. Immunostaining and Western blot analysis were used to measure cytokeratin K3 and K12 expression with AE5 and AK12 antibodies. Karyotype analysis was performed based on comparison of the RCE chromosomal complement with its parent tissue. EGF receptor activation was evaluated based on immunochemistry and Western blot analyses of EGF receptor dimerization and phosphorylation. Functional status of EGF receptor was determined through measurements of EGF-induced stimulation of ERK-2 activity, which is a component of the mitogen-activated protein kinase cascade (MAPK). This was done by immunocomplex and kinase assay using anti-ERK antibodies and a specific substrate. EGF-induced increases in proliferation and cell cycle progression were determined based on measurements of [(3)H]-thymidine incorporation, G(2)-specific cyclin B1 expression and cell cycle mapping.. From days 7 to 14, K12 expression increased based on marked rises in the levels of a 55 kD band. At day 14, a 64 kD band also appeared indicative of K3 expression. Karyotype analysis showed that there were no chromosomal losses due to SV-40 transformation. Upon exposure to EGF (5 ng/ml) for 1 min, EGF receptors were activated and formed clusters indicating that autophosphorylation and multimerization of the EGF receptor were occurred. In the presence of serum growth factors or EGF, ERK-2 kinase activity was markedly increased with a bell-shaped time-dependent activation pattern. Cell cycle progression was analyzed in G(1)/S boundary synchronized RCE cells. After releasing the cells into modified Supplemented Hormonal Epithelium Medium containing 10% serum and DMEM/F-12 medium, 80% of the cells had entered the S phase within 2 h. In addition, time dependent changes in [(3)H]-hymidine incorporation over 8 h confirmed RCE passage through the G(1)/S checkpoint. There were more RCE cells entered the G(2)/M phase of cell cycle in the 6-8 h interval after their release. Another indication of cell cycle progression into the G(2)/M phase was that at 8-10 h cyclin B(1) expression reached its maximal level.. RCE in passage number 12-20 are a physiologically relevant model for studies on growth factor receptor mediated control of cell cycle progression and differentiation in its parent tissue as each of these phenomena were conserved: 1) EGF-induced EGF receptor activation; 2) EGF-activated ERK signaling; 3) expression of cornea-specific differentiation markers; 4) karyotype profile; and 5) cell cycle control and progression.

Topics: Animals; Blotting, Western; Cell Differentiation; Cell Division; Cell Line, Transformed; Cell Transformation, Viral; Cyclin B; Cyclin B1; DNA; Epidermal Growth Factor; Epithelium, Corneal; ErbB Receptors; Fluorescent Antibody Technique, Indirect; Karyotyping; Keratins; Mitogen-Activated Protein Kinase 1; Phosphorylation; Rabbits; Signal Transduction; Simian virus 40

The tumor suppressor transcription factor IRF-1 inhibits cell growth. In this report we show that IRF-1 also induces apoptosis of highly transformed and tumorigenic cell lines. This activity of IRF-1 is demonstrated with cell lines expressing HER oncogenes and an activatable IRF-1 fusion protein. Growth of cell lines expressing inactive HER1 is inhibited on IRF-1 activation. In contrast, the same cells are killed by apoptosis when HER1 and IRF-1 are activated simultaneously. We identified promoters stimulated synergistically by IRF-1 and by activated HER1. To determine the signals causing transcriptional synergism and/or apoptosis we tried to modulate these effects by various dominant negative acting proteins. Dominant negative STAT5alpha abolished both induction of apoptosis and transcriptional synergy of IRF-1 and HER. Thus, these results provide new insights into the mechanism of oncogene-dependent apoptosis induced by the activation of a tumor suppressor.

Topics: 3T3 Cells; Adenovirus E1A Proteins; Adenovirus E1B Proteins; Animals; Cell Line, Transformed; Cell Transformation, Viral; DNA-Binding Proteins; Epidermal Growth Factor; ErbB Receptors; Estradiol; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Humans; Interferon Regulatory Factor-1; Interferons; Mice; Milk Proteins; Moloney murine leukemia virus; Oncogenes; Phosphoproteins; Promoter Regions, Genetic; ras Proteins; Receptor, ErbB-2; Recombinant Fusion Proteins; Sarcoma; Signal Transduction; STAT5 Transcription Factor; Trans-Activators; Transcription, Genetic; Transfection

EBV-LMP gene transfection, radioimmuno-binding assay (RIA) and colorimetric cell viability assay (MTT) were used to investigate the effect and relationship of EBV-LMP and EGF autocrine on the growth and proliferation of well differentiated nasopharyngeal carcinoma cell line (CNE1). Autocrine EGF was detected in the supernant of CNE1 cells and CNE1 cells could grow in serum-free medium, implicating that the cell proliferation-promoting effect of EGF autocrine was present in the CNE1 cell line. Nevertheless the positive signals of LMP gene polymerase chain reaction and LMP Mab immunohistochemical staining in pCMV alpha-LMP DNA transfected cells, confirmed the successful transfection of LMP gene. EGF autocrine amount and the proliferation of CNE1 cells in serum-free medium were more obvious in post-transfected cells than those in untransfected cells. The present study represents the first report on promotion of EGF autocrine in EBV-LMP-transfected cells, thus promoting cell proliferation.

Topics: Autocrine Communication; Cell Transformation, Viral; Epidermal Growth Factor; Herpesvirus 4, Human; Humans; Nasopharyngeal Neoplasms; Oncogene Proteins, Viral; Tumor Cells, Cultured; Viral Matrix Proteins

Growth factor receptors may be transactivated not only by homologous receptors, but also by heterologous receptors. We have investigated this possibility, using for this purpose R-/EGFR cells, which are mouse embryo cells devoid of IGF-I receptors, but overexpressing the EGF receptor. At variance with mouse embryo cells with a wild-type number of IGF-I receptors and overexpressing the EGF receptor, R-/EGFR cells cannot grow in EGF only, nor can they form colonies in soft agar. However, if a wild type human IGF-I receptor is stably transfected into R-/EGFR cells, growth in EGF and colony formation in soft agar are restored. To determine a possible interaction between the two receptors, we transfected into R-/EGFR cells a number of IGF-I receptor mutants with different impaired functions. The only IGF-I receptor that cannot reverse the growth phenotype of R-/EGFR cells is a receptor with a point mutation at the ATP-binding site. All other mutant receptors, even when incapable of responding to IGF-I with a mitogenic signal, made R-/EGFR cells fully capable of responding with growth to EGF stimulation. IGF-I receptor mutants that are mitogenic but not transforming made R-/EGFR cells grow in EGF only, but were incapable of inducing the transformed phenotype. The mutant IGF-I receptors are activated (tyrosyl phosphorylation of IRS-I) in response to EGF. These experiments indicate that certain IGF-I receptor mutants with loss of function can be reactivated intracellularly by an overexpressed EGF receptor and confirm that the C-terminus of the IGF-IR is required for its transforming activity.

Topics: Adenosine Triphosphate; Animals; Binding Sites; Cell Division; Cell Line; Cell Transformation, Viral; Epidermal Growth Factor; ErbB Receptors; Humans; Insulin-Like Growth Factor I; Mice; Mutation; Phenotype; Phosphorylation; Protein-Tyrosine Kinases; Receptor, IGF Type 1; Transcriptional Activation

The ATDC gene was originally identified by its ability to complement the radiosensitivity defect of an ataxia telangiectasia (AT) fibroblast cell line. Because hypersensitivity to ionizing radiation is an important feature of the AT phenotype, we reasoned that ATDC may function generally in the suppression of radiosensitivity. Previous work in our laboratory focused on radiosensitization mechanisms in human squamous carcinoma (SC) cells, especially A431 cells. To establish a basis for investigating the role of ATDC in radiation-responsive signaling pathways in human SC cells, we characterized ATDC message and protein expressions in A431 cells. ATDC message expression was also compared among human epidermoid cells (A431 cells, HaCaT spontaneously immortalized human keratinocytes and normal human epidermal keratinocytes) and a normal human fibroblast cell line (LM217). We made the following major observations: (i) the relative abundance of ATDC message is substantially higher in the epidermoid cells than in the fibroblast cell line, which has a message level comparable to those reported for other fibroblast lines; (ii) ATDC is constitutively phosphorylated on serine/threonine in A431 cells; (iii) in A431 cells, ATDC is a substrate for the serine/threonine protein kinase C (PKC) but not the epidermal growth factor (EGF) receptor tyrosine kinase; and (iv) EGF decreases ATDC message and protein expressions in A431 cells after a 24-hr exposure. The phosphorylation studies suggest that the ability of ATDC to modulate cellular radiosensitivity may be mediated in part through a PKC signaling pathway.

Topics: Ataxia Telangiectasia; Base Sequence; Carcinoma, Squamous Cell; Cell Line, Transformed; Cell Transformation, Viral; DNA-Binding Proteins; DNA, Complementary; Epidermal Growth Factor; Fibroblasts; G1 Phase; Gene Expression Regulation, Neoplastic; Humans; Keratinocytes; Molecular Sequence Data; Neoplasm Proteins; Phosphorylation; Protein Kinase C; Protein Processing, Post-Translational; Recombinant Fusion Proteins; Recombinant Proteins; RNA, Messenger; RNA, Neoplasm; Simian virus 40; Skin; Transcription Factors; Tumor Cells, Cultured

Epiderstatin, a distinctive glutarimide antibiotic isolated from Streptomyces pulveraceus subsp. epiderstagenes, has been revealed to be a potent inhibitor of the signal transduction of epidermal growth factor (EGF). Epiderstatin inhibited the DNA synthesis induced by various peptide growth factors in a mouse epidermal cell line, BALB/MK, without inhibiting protein tyrosine kinase activity of EGF-receptor or serine/threonine kinase activity of protein kinase C. The 50% inhibitory concentration (IC50) value of epiderstatin for the EGF-stimulated incorporation of [3H]thymidine into BALB/MK cells was about 10 nM. When epiderstatin was added to the quiescent cells simultaneously with EGF-stimulation, the cells did not reenter into the growing cell cycle. The action of epiderstatin proceeded from the overexpression of c-fos and the suppression of c-myc transcription when EGF was added to quiescent BALB/MK cells.

Topics: Actins; Animals; Cell Cycle; Cell Division; Cell Transformation, Viral; DNA; Epidermal Cells; Epidermal Growth Factor; Epidermis; ErbB Receptors; Gene Expression Regulation; Genes, fos; Genes, myc; Keratinocytes; Mice; Mice, Inbred BALB C; Piperidones; Protein Kinases; Protein-Tyrosine Kinases; Pyridones; Signal Transduction; Stimulation, Chemical; Transcription, Genetic

A benzodiazepine peptidomimetic, BZA-5B, inhibits farnesylation of H-Ras and normalizes the morphology of Rat-1 cells transformed with H-RasV12 at concentrations that do not affect the growth of untransformed Rat-1 cells. In the current experiments, we show that BZA-5B decreases the active forms of enzymes in the mitogen-activated protein (MAP) kinase signaling cascade, including Raf, MAP kinase kinase (MEK), and MAP kinase, in cells transformed with H-RasV12. BZA-5B had no effect on these enzymes in cells transformed with H-RasV12,L189, which is geranylgeranylated rather than farnesylated. In cells transformed with H-RasV12, BZA-5B reduced the activities of enzymes in the MAP kinase pathway at concentrations that only partially blocked farnesylation of H-RasV12, suggesting that nonfarnesylated H-RasV12 is a dominant inhibitor of the action of farnesylated H-RasV12 in the BZA-5B treated cells. In untransformed Rat-1 cells, BZA-5B did not inhibit MAP kinase activity nor did it prevent the acute activation triggered by epidermal growth factor, even though farnesylated endogenous H-Ras was no longer detectable. These data raise the possibility that untransformed cells contain a form of Ras (K-Ras or N-Ras) whose prenylation is not inhibited by BZA-5B, thus allowing them to resist the effects of BZA-5B.

Topics: Animals; Benzodiazepines; Calcium-Calmodulin-Dependent Protein Kinases; Cell Division; Cell Line; Cell Transformation, Viral; Cells, Cultured; Enzyme Activation; Epidermal Growth Factor; In Vitro Techniques; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Oligopeptides; Oncogene Protein p21(ras); Phosphorylation; Protein Prenylation; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-raf; Proto-Oncogene Proteins p21(ras); Rats; Signal Transduction; Transfection

In the present study, we examine the effects of all-trans-retinoic acid (RA) and interferons-alpha and -gamma (IFN-alpha and IFN-gamma) on the growth of HPV16-immortalized cell lines, ECE16-1 and CaSki. Treating proliferating ECE16-1 cells with RA causes a concentration-dependent decrease in cell number. At 1 microM RA, cell growth is suppressed by 65% and the level of mRNA encoding cytokeratin K5, a biochemical marker of retinoid action, is also suppressed. In contrast, the level of transcript encoding the HPV16 oncogenes, E6 and E7, is reduced by only 5 to 10%. IFN-alpha at 1000 IU/ml or IFN-gamma at 200 IU/ml suppresses growth by 70%. This growth suppression by IFN-gamma is correlated with a > 90% reduction in E6/E7 mRNA levels. Additional growth suppression is observed upon simultaneous treatment with retinoid and interferon. Optimal suppression is observed in the presence of 200 IU/ml IFN-gamma and 1 microM RA. The rank order of effectiveness is IFN-gamma/RA > IFN-alpha/RA = IFN-gamma > RA > IFN-alpha. In contrast to the suppression of ECE16-1 cell growth, RA causes a concentration-dependent increase in CaSki cell number (50-60%) which is optimal at 1 microM RA. Cytokeratin K5 mRNA levels are markedly suppressed, and E6/E7 mRNA levels increased by 5% under these conditions. IFN-alpha at 1000 IU/ml or IFN-gamma at 200 IU/ml decreases CaSki cell growth by 20 and 45%, respectively, and 200 IU/ml of IFN-gamma reduce E6/E7 expression to undetectable levels. Addition of RA (1 microM) partially counters the IFN-dependent suppression of growth and E6/E7 mRNA levels. Our results suggest that retinoid-dependent changes in human papillomavirus-immortalized cervical cell proliferation are not always correlated with changes in E6/E7 transcript levels.

Topics: Animals; Cell Division; Cell Line; Cell Line, Transformed; Cell Transformation, Viral; Cervix Uteri; Dose-Response Relationship, Drug; Epidermal Growth Factor; Epithelial Cells; Epithelium; Female; Gene Expression; Humans; Interferon-alpha; Interferon-gamma; Kinetics; Mice; Mice, Nude; Oncogenes; Papillomaviridae; RNA, Messenger; Transcription, Genetic; Transplantation, Heterologous; Tretinoin; Uterine Cervical Neoplasms

The effect of SV40 transformation on the mitogenic responsiveness of Balb/c-3T3 cells to platelet-derived growth factor was examined. The SV40 transformed cell line (SV40-3T3) did not require exogenous PDGF for growth in a serum-free medium containing insulin, transferrin and high density lipoprotein as macromolecular supplements. The SV40-3T3 cells were found to have undetectable PDGF-stimulated PDGF receptor autophosphorylation and markedly reduced PDGF binding. PDGF-induced PDGF receptor autophosphorylation and PDGF binding of the normal 3T3 cells were not reduced by incubation with conditioned medium from SV40 transformed cells; nor could PDGF binding to SV40-3T3 be increased by an 8-hour incubation with 0.5 mM suramin, a compound known to prevent the binding of PDGF to its receptor. These data suggest that SV40-3T3 cells do not produce PDGF in quantities that are sufficient to down regulate their PDGF receptor and suggest that the reduced PDGF responsiveness and PDGF receptor number in SV40-3T3 cells may be a reflection of a more permanent, perhaps genetic, alteration.

Topics: 3T3 Cells; Animals; Cell Transformation, Viral; Culture Media, Serum-Free; Down-Regulation; Epidermal Growth Factor; Growth; Mice; Mice, Inbred BALB C; Mitogens; Phosphorylation; Platelet-Derived Growth Factor; Receptors, Platelet-Derived Growth Factor; Simian virus 40; Suramin

Topics: Adult; Antigens, Polyomavirus Transforming; Breast; Cell Line, Transformed; Cell Transformation, Viral; Cellular Senescence; Epidermal Growth Factor; Female; Humans; Middle Aged; Stem Cells

Although human papillomavirus type 16 (HPV-16) is believed to be a major etiological agent in the development of cervical cancer, the biological function of several of its early genes remains to be established. In the present study, we have defined some of the biological properties of the E5 gene from HPV-16. Expression of the HPV-16 E5 gene in 3T3-A31 cells induced transformation to anchorage-independent growth (colony formation in soft agar). Addition of epidermal growth factor (EGF) to the soft-agar medium caused the E5-expressing cells to form larger colonies than those formed in the absence of EGF. Parental 3T3-A31 cells did not form colonies in soft agar either in the presence or in the absence of EGF. Analysis of clones expressing high levels of E5 mRNA revealed that these cells also expressed higher levels of c-fos mRNA in response to serum, EGF and platelet-derived growth factor (PDGF) than did the parental 3T3-A31 cells. Cells expressing the E5 gene were also capable of accelerated growth in low serum and were more tumorigenic in nude mice than were control cells. We conclude that the E5 gene from HPV-16 is an oncogene which transforms cells in part through enhancing signal transduction from growth factors to the nucleus.

Topics: 3T3 Cells; Animals; Carcinogenicity Tests; Cell Division; Cell Nucleus; Cell Transformation, Viral; Colony-Forming Units Assay; Epidermal Growth Factor; Gene Expression Regulation, Viral; Genes, fos; Genes, Viral; Humans; Mice; Mice, Nude; Oncogene Proteins v-fos; Oncogenes; Papillomaviridae; RNA, Messenger; Signal Transduction; Transfection

We have until recently made several unsuccessful attempts to assign any activity to the human papillomavirus type 16 (HPV-16) E5 gene product. However, studies with the bovine papilloma virus 1 (BPV-1) E5 protein indicated an interaction with the epidermal growth factor receptor (EGFR). In light of the overall similarity between the HPV and BPV E5 proteins we attempted to determine whether the HPV-16 E5 gene had any common activity. In cells expressing high levels of EGFR plus HPV-16 E5 we found a dramatically increased proliferative activity in soft-agar assays in the presence of EGF. The specificity of this activity was monitored by the addition of other mitogenic agents. The phorbol ester phorbol 12-myristate 13-acetate (PMA) had no effect on the E5-containing cells, although insulin weakly stimulated their growth in soft agar. Further analysis revealed the same number of EGF receptors were present on the E5-containing cells as on the control cells, although the E5 cells were more sensitive to lower concentrations of EGF. These results imply that E5 is amplifying the mitogenic signals from the EGFR in an as yet unknown manner, but which may form the basis of interactions with a variety of growth factor receptors. This report brings to three the number of transforming genes encoded by HPV-16.

Topics: 3T3 Cells; Animals; Bovine papillomavirus 1; Cell Transformation, Viral; Colony-Forming Units Assay; Drug Interactions; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Viral; Genes, Viral; Humans; Mice; Papillomaviridae; Transfection

To study the effect of SV40 T-antigen in mammary epithelial cells, a rat beta-casein promoter-driven SV40 early-region construct was stably introduced into the clonal mouse mammary epithelial cell line HC11. With the expression of the viral T-antigens under the control of a hormone-inducible promoter, it was possible to dissociate the effects of different levels of T-antigen expression on cell growth, morphology, and gene expression. Following hormonal induction, a rapid but transient induction of T-antigen was observed, followed by a delayed induction of H4 histone mRNA. In T-antigen-positive HC11 cells cultured in the absence of EGF, the expression of basal levels of T-antigen (in the absence of hormonal induction) led to a decreased doubling time and an increased cell density. In the presence of EGF, T-antigen expression resulted additionally in an altered cell morphology. Despite the effects of T-antigen on cell growth and gene expression, the cells were unable to form colonies in soft agar and were nontumorigenic when transplanted into cleared mammary fat pads. They were, however, weakly tumorigenic in nude mice. Relatively high levels of p53 protein synthesis were observed in both the transfected HC11 cells and the parental COMMA-D cells, as compared to 3T3E fibroblasts and another mammary epithelial cell line. The HC11 and COMMA-D cells synthesized approximately equal levels of wild-type and mutated p53 proteins as defined by their reactivities with monoclonal antibodies PAb246 and PAb240, respectively. Interactions between excess p53 and T-antigen may, in part, explain the failure of these cells to display a completely transformed phenotype.

Topics: Adenovirus Early Proteins; Animals; Antigens, Polyomavirus Transforming; Caseins; Cell Division; Cell Transformation, Viral; Clone Cells; Epidermal Growth Factor; Epithelial Cells; Female; Gene Expression; Genes, Viral; Histones; Hydrocortisone; Mammary Glands, Animal; Mice; Oncogene Proteins, Viral; Plasmids; Prolactin; RNA, Messenger; Simian virus 40; Trans-Activators; Transfection

Epiderstatin is a unique glutarimide antibiotic which was found by screening for inhibitors of the signal transduction of epidermal growth factor (EGF). The antibiotic (0.01 microM) was found to reverse the morphology of NRK cells that were infected with a temperature-sensitive mutant of Rous sarcoma virus (srcts-NRK) from the transformed phenotype to the normal phenotype at the permissive temperature (32 degrees C). Epiderstatin did not inhibit the protein kinase activity of p60v-src. The cell cycle progression of src(ts)-NRK cells was blocked at G0/G1 phase, which was caused by the inhibition of biosynthesis of p60v-src but not the transcription of v-src mRNA.

Topics: Animals; Antibiotics, Antineoplastic; Avian Sarcoma Viruses; Biotechnology; Cell Cycle; Cell Division; Cell Line, Transformed; Cell Transformation, Viral; Epidermal Growth Factor; Mutation; Oncogene Protein pp60(v-src); Phenotype; Piperidones; Pyridones; Rats; RNA, Messenger; Temperature

Epidermal growth factor (EGF)-induced down-regulation of its receptor is an obligatory pathway for cellular regulation of EGF-specific receptor (EGF-R) in normal and malignant cells. BNER4 cells are mouse Balb/3T3 cells transfected with the human EGF-R complementary DNA (cDNA). Polyoma middle T antigen-transfectants of BNER4, B4/MT-2, B4/MT-13, B4/MT-23, and B4/MT-24, showed diminished down-regulation of cell surface human EGF-R in response to EGF relative to the parental BNER4 cells. Also, the v-src-transfectants B4/SRC-13 and B4/SRC-24 showed much less down-regulation than BNER4 cells, whereas H-ras-transfectants of BNER4, B4/RAS-24 and B4/RAS-25, showed EGF-induced down-regulation of the cell surface EGF-R similar to that of BNER4. EGF induced DNA synthesis more than 20-fold in BNER4, but induced only about a 1.5- to 6-fold increase in the middle T antigen- and v-src-transfectants. EGF-Rs of the middle T antigen-transfectants were metabolically stable in the presence of EGF in comparison with their parental BNER4 cells. EGF-Rs of BNER4 cells degraded with half-lives of about 2 h in the presence of EGF, but those of the middle T antigen transformants were found to be highly stabilized in the presence of EGF. On the other hand, transfection with polyoma middle T antigen (MTAg) cDNA causes malignant transformation of Balb/3T3 cells, but not its monensin (an ionophoric antibiotic)-resistant mutant MO-5 cells, which have no significant EGF binding activity. Transfection of human EGF-R cDNA into MO-5 leads to the expression of high levels of human EGF-R in MNER31. Unlike the polyoma MTAg transfectants of BNER4, EGF-R in polyoma MTAg cDNA-transfectants into MNER31, M31/MT-13 and M31/MT-14, were down-regulated to levels similar to those of their parental MNER31. Exposure to EGF induced a more than 10-fold increase in DNA synthesis of quiescent BNER4, MNER31, M31/MT-13, and M31/MT-14 cells. Polyoma middle T antigen or v-src appears to modulate EGF-induced down-regulation of EGF-R, possibly through interaction of the receptor with the viral oncogenes, and this interaction may be altered in the mutant.

Topics: 3T3 Cells; Animals; Antigens, Polyomavirus Transforming; Base Sequence; Cell Division; Cell Transformation, Neoplastic; Cell Transformation, Viral; DNA; Down-Regulation; Drug Resistance; Epidermal Growth Factor; ErbB Receptors; Genes, src; Mice; Molecular Sequence Data; Monensin; Mutation; Phosphorylation; Transfection

The effect of interferon-gamma (IFN-gamma) on epidermal growth factor (EGF) receptor binding and the proliferation of normal and simian virus 40 (SV40)-transformed human fibroblast cells was compared under identical culture conditions. IFN-gamma induced an enhancement of EGF binding to normal cells, whereas it decreased the EGF binding to SV40-transformed cells. Half-maximal enhancement occurred at 72 h after the normal cells were exposed to 10 U/ml of IFN-gamma, and maximal stimulation was obtained at about 10(2) U/ml of IFN-gamma at 72 h. On the other hand, half-maximal reduction was observed for SV40-transformed cells at less than 10 U/ml of IFN-gamma at 72 h, and maximal reduction was obtained at around 10(3) U/ml of IFN-gamma at 72 h. Scatchard analysis indicated that the number of EGF binding sites of normal and SV40-transformed cells was calculated to be 1.6 x 10(5) and 0.88 x 10(5) per cell, respectively, and was little altered by IFN-gamma treatment. The dissociation constant (Kd) of normal cells, however, decreased from 4.5 nM (control) to 2.0 nM (IFN-gamma-treated), while the Kd of SV40-transformed cells increased from 3.6 nM (control) to 17.0 nM (IFN-gamma-treated). The immunoprecipitation of 125I-labeled EGF-bound EGF receptors with anti-receptor antiserum indicated that a 72-h IFN-gamma treatment did not induce a conformational alteration in the EGF receptors of both normal and transformed cells. The DNA synthesis of normal cells was enhanced by EGF, and IFN-gamma treatment potentiated the effect of EGF on DNA synthesis, probably due to the increased binding affinity of EGF to the cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Cell Transformation, Viral; Cells, Cultured; DNA; Epidermal Growth Factor; ErbB Receptors; Fibroblasts; Humans; Interferon-gamma; Precipitin Tests; Reference Values; Thymidine

The mechanisms and consequences of persistence of non-transforming viruses are poorly understood. Reovirus infections are usually regarded as cytocidal and infection is associated with inhibition of cellular protein and DNA synthesis. Reovirus infection of the polarized epithelial MDCK cell line is not associated with inhibition of protein synthesis, and cells become persistently infected and continue to grow without c.p.e. after infection. After several passages, virus persistence is associated with profound morphological and functional changes. The cells lose their usual cobblestone appearance and acquire a fibroblastic, undifferentiated morphology. This is associated with an inability to form tight junctions. In addition, expression of epidermal growth factor receptors and one adhesion protein is altered in the persistently infected cells. These results demonstrate that reovirus persistence will occur readily, and that infection of differentiated cells with a non-transforming virus can lead to loss of differentiation and abnormal protein expression.

Topics: Animals; Cell Line; Cell Transformation, Viral; Dogs; Electrophoresis, Polyacrylamide Gel; Epidermal Growth Factor; Epithelial Cells; Epithelium; Kidney; Reoviridae; RNA, Viral; Serial Passage; Viral Plaque Assay

In a previously published report (Exp. Cell. Res. 161:421 (1985] we have demonstrated that cultured normal and transformed keratinocytes exhibit two classes of EGF binding sites after growth under normal Ca2+ conditions but only low-affinity binding sites after growth under low Ca2+ conditions. Here we demonstrate the presence of high-affinity binding sites in transformed keratinocytes grown under low Ca2+ conditions, using a specific monoclonal anti EGF-receptor antibody.

Topics: Antibodies, Monoclonal; Calcium; Carcinoma, Squamous Cell; Cell Division; Cell Transformation, Viral; Cells, Cultured; Epidermal Growth Factor; ErbB Receptors; Humans; Keratinocytes; Simian virus 40; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

A serum-free culture system was used to compare the nutritional requirements of mouse mammary cells transformed by bovine papillomavirus type 1 (ID13 cells) and the uninfected parent line (C127 cells). The serum-free, chemically defined medium used for this study was an MCDB 151-based medium (MCDB 151+S+I), supplemented with epidermal growth factor, transferrin, hydrocortisone, ethanolamine, phosphoethanolamine, retinoic acid, trace metals, and insulin. Proliferation of either cell type in serum-free culture required the addition of 250 micrograms/ml of insulin. ID13 cells have a doubling time of greater than 96 h in MCDB 151+S+I, whereas C127 cells have a doubling time of 60 h. This is in sharp contrast to the growth characteristics of the two cell types in 10% fetal bovine serum, where doubling times for the ID13 and C127 cells are 24 and 30 h, respectively. Culture of the cells in a serum-free medium has therefore revealed that the papillomavirus-transformed cells have more stringent growth requirement than the uninfected parent line.

Topics: Animals; Cell Line, Transformed; Cell Transformation, Viral; Culture Media; Dose-Response Relationship, Drug; Epidermal Growth Factor; Ethanolamines; Female; Hydrocortisone; Insulin; Mammary Glands, Animal; Metals; Mice; Nutritional Requirements; Papillomaviridae; Transferrin; Tretinoin

Nucleocytoplasmic transport of fluorescent-labeled macromolecules was investigated in transformed and nontransformed 3T3 fibroblasts. Insulin and epidermal growth factor enhanced transport three-fold after 1-2-h incubation with nontransformed adhering fibroblasts; no enhancement of transport was observed for spherical unattached fibroblasts. The concentration of growth factor for maximal enhancement was 3-10 nM. Nuclear transport for Kirsten murine sarcoma virus-transformed BALB/c 3T3 fibroblasts, however, was maximally enhanced before addition of growth factors; addition of insulin or epidermal growth factor causes no additional transport enhancement. Transformation also minimizes cell shape effects on macromolecular nuclear transport. These results provide evidence that protein growth factors and oncogenic transformation may use a similar mechanism for activation of nuclear transport.

Topics: Animals; Biological Transport; Cell Line; Cell Nucleus; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cytoplasm; Dextrans; Diffusion; Epidermal Growth Factor; Growth Substances; Insulin; Mice; Nuclear Envelope; Receptors, Cell Surface; Spectrometry, Fluorescence

Topics: Adult; Aminoisobutyric Acids; Biological Transport, Active; Carcinoma, Squamous Cell; Cell Line; Cell Transformation, Viral; Cells, Cultured; Epidermal Cells; Epidermal Growth Factor; Epidermis; Humans; Infant, Newborn; Insulin; Kinetics; Simian virus 40; Sodium; Tumor Cells, Cultured

Variants (G2, G5) resistant to the cancer chemotherapeutic drug methylglyoxal bis (guanylhydrazone) (MGBG) were isolated from adenovirus type 2 transformed rat brain cells (F4; Sircar et al., 1987). Although at least one of these variants continued to express the adenovirus Ela and Elb transforming proteins, they both exhibited a detransformed phenotype as witnessed by flat morphology, loss of anchorage independent growth, and tumor forming capacity. Reverse transformation suggested the possibility of changes in growth factor receptors and the production of transforming growth factors. To test this possibility, we investigated the status of epidermal growth factor receptors (EGF-r) and transforming growth factor alpha (TGF-alpha) production in F4, G2 and G5 cells. The level of 125I-labeled EGF binding to intact drug resistant cells increased by 2- to 3-fold compared to the transformed parental cell. Scatchard analysis suggests that increased binding was the result of increased receptor levels rather than altered affinity of receptor for ligand. The production of growth factors which compete with 125I-labeled EGF binding declined in the detransformed G2 and G5 cells to a level intermediate between transformed (F4) and normal cells (FR3T3). EGF-receptor increase and the complementary decrease in growth factor production in the drug resistant variants may be associated with detransformation.

Topics: Adenoviridae; Animals; Cell Line, Transformed; Cell Transformation, Viral; Drug Resistance; Epidermal Growth Factor; ErbB Receptors; Growth Substances; Mitoguazone; Peptide Biosynthesis; Phenotype; Transforming Growth Factors

The transmembrane potential of Rous sarcoma virus (RSV)-infected Rat-1 cells, expressing the pp60v-src protein kinase, is markedly less negative (by approximately 30 mV) than that of their normal counterparts. By contrast, the membrane potential of Rat-1 cells infected with Kirsten sarcoma virus is virtually unaltered. The RSV-induced membrane depolarization is shown to be due to a severalfold increase in the cation permeability ratio (PNa/PK) of the plasma membrane. When cells infected with a temperature-sensitive mutant of RSV (ts LA29), encoding a src protein with heat-labile kinase activity, are shifted from the nonpermissive to the permissive temperature, a rapid and sustained membrane depolarization is observed. Conversely, thermal inactivation of the ts LA29 pp60v-src kinase activity rapidly restores the membrane potential to near normal levels. Addition of epidermal growth factor, platelet-derived growth factor, or insulin to uninfected cells fails to cause a detectable change in membrane potential. We conclude that, unlike growth factor receptor tyrosine kinases, pp60v-src can induce, either directly or indirectly, a major change in the membrane permeability to monovalent cations.

Topics: Animals; Avian Sarcoma Viruses; Cell Membrane Permeability; Cell Transformation, Viral; Epidermal Growth Factor; Hot Temperature; Insulin; Kirsten murine sarcoma virus; Mathematics; Membrane Potentials; Oncogene Protein pp60(v-src); Platelet-Derived Growth Factor; Protein Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins pp60(c-src); Rats; Retroviridae Proteins; Temperature

We have analyzed the relationship between expression of the transformed phenotype and thyroid hormone (triiodothyronine, T3) inducibility of Na,K-ATPase and binding of 125I-epidermal growth factor (EGF) to cell membrane receptors in wild-type (wt) and mutant type 5 adenovirus (Ad5)-transformed CREF cells displaying a cold-sensitive (cs) expression of the transformed phenotype. CREF cells respond to thyroid hormone treatment with increased Na,K-ATPase activity and bind similar levels of 125I-EGF at 32 degrees C, 37 degrees C and 39.5 degrees C. In contrast, CREF cells transformed by wt Ad5 or the E1a plus E1b-transforming genes of wt Ad5 are refractile to T3 treatment and bind lower levels of 125I-EGF than CREF cells at all three temperatures. By employing a series of cloned CREF cell lines transformed by a host-range cold-sensitive mutant virus, H5hr1 or H5dl101, or the E1a or E1a plus E1b genes from these viruses, we have investigated expression of the transformed state and its relationship with hormone inducibility and EGF binding. When cs virus, cs E1a- or cs E1a plus E1b-transformed CREF clones were grown at 32 degrees C, a nonpermissive transforming temperature in which cs-transformed cells exhibit properties similar to untransformed CREF cells, T3 induced Na,K-ATPase activity and these cells bound similar levels of 125I-EGF as CREF cells. However, when cs virus- and cs Ela plus E1b-transformed CREF clones were incubated at 37 degrees C or 39.5 degrees C, temperatures at which cs-transformed cells exhibit properties similar to wt Ad5-transformed CREF cells, they did not respond to T3 and bound lower levels of 125I-EGF than CREF cells. In the case of cs E1a-transformed CREF clones, thyroid hormone responsiveness was observed at both 32 degrees C and 37 degrees C, but not at 39.5 degrees C. By performing temperature shift experiments--i.e. 32 degrees C to 37 degrees C, 32 degrees C to 39.5 degrees C, 37 degrees C to 32 degrees C, and 39.5 degrees C to 32 degrees C, it was demonstrated that after a shift from lower to higher temperature a 24-hr lag period was required for cs-transformed CREF cells to lose T3 inducibility and exhibit reduced EGF binding, whereas 96 hr after a shift from higher to lower temperature a 96-hr lag period was required for cs-transformed cells to regain T3 inducibility and increased 125I-EGF binding.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Adenoviridae; Animals; Cell Cycle; Cell Line, Transformed; Cell Transformation, Viral; Cold Temperature; Enzyme Induction; Epidermal Growth Factor; Fibroblasts; Kinetics; Mutation; Sodium-Potassium-Exchanging ATPase; Temperature; Time Factors; Triiodothyronine

Adenovirus type 12 (Ad12) Ela-transformed rat cells (HY1) grew in methocel medium containing 9% fetal calf serum (FCS), but the frequency of colony formation was very low (the order of 10(-4)). The addition of conditioned medium or a high concentration of serum (20% FCS) to the methocel medium accelerated colony formation, and plating efficiency increased 10- to 100-fold. In contrast, in stationary culture, HY1 cells grew well even in 1%-FCS medium. These results indicate that HY1 cells require high concentrations of growth factors for anchorage-independent growth. The effects of conditioned medium or FCS also were demonstrated in several transformed cell lines induced by transfection of combined sets of Ad12-transforming genes (E1a, E1b and E4). These growth behaviors suggest that the first step in cell transformation with adenovirus 12 is the acquisition of responsiveness to growth factors in methocel culture, which must be the function of the Ad12-E1a gene products. The function of the other two Ad12-transforming genes was discussed.

Topics: Adenovirus Early Proteins; Adenoviruses, Human; Animals; Cell Adhesion; Cell Cycle; Cell Transformation, Viral; Culture Media; Epidermal Growth Factor; Fibronectins; Genes, Viral; Growth Substances; Oncogene Proteins, Viral; Rats

The effects of src oncogene expression on epidermal growth factor (EGF) receptors have been investigated in mouse 3T3 and rat-1 fibroblasts. Transformation of both cell types with src resulted in marked reductions in cellular EGF receptor levels, as assayed by either 125I-EGF binding or immunoprecipitation of receptor protein from radiolabeled cell lysates. In contrast to cells transformed by other types of retroviral oncogenes, the loss of EGF receptors in the src-transformed cells did not appear to be due to secreted transforming growth factor-alpha (TGF-alpha), since such factors were undetectable in culture fluids from the src-transformed cells. By several criteria of transformation, an EGF-receptorless cell line infected with src was shown to be transformed, suggesting that EGF receptors themselves are not obligatory to the src transformation process. We suggest that pp60src down-modulates EGF receptors by an intracellular mechanism and that the loss of the receptors is symptomatic of more general effects of pp60src on the machinery of growth regulation.

Topics: Animals; Avian Sarcoma Viruses; Cell Line; Cell Transformation, Viral; Culture Media; Epidermal Growth Factor; ErbB Receptors; Fibroblasts; Mice; Oncogene Protein pp60(v-src); Peptides; Protein-Tyrosine Kinases; Rats; Retroviridae Proteins; Transforming Growth Factors

The effects of retinoic acid on the epidermal growth factor (EGF) receptor binding and cell growth of normal and simian virus 40 (SV40)-transformed BALB/c 3T3 cells were compared under identical culture conditions. Retinoic acid induced a rapid enhancement of EGF binding to SV40-transformed cells. Half-maximal enhancement occurred at about 7 h after the cells were exposed to 20 ng/ml of retinoic acid, and maximal stimulation (from 2.5- to 3.5-fold over the control) was obtained after 12 h of exposure. The kd of the control and retinoic acid-treated cells was calculated to be 8.0 X 10(-10) M and 8.2 X -10 M, respectively. However, the number of unoccupied EGF binding sites increased from 0.98 X 10(4) to 2.28 X 10(4) per cell. Normal 3T3 cells would not respond to retinoic acid unless they were cultured in serum-containing medium. After 96 h of exposure, only a 50% enhancement of EGF binding was observed. The EGF receptor number of the untreated normal cells was calculated to be 1.82 X 10(4) per cell, twice the number expressed by untreated SV40-transformed cells. The increase of EGF receptor number caused by retinoic acid in SV40-transformed cells was blocked by either actinomycin D or cycloheximide treatment. These results indicated that SV40 transformation of BALB/c 3T3 cells altered the regulatory mechanism governing the complement of cell surface EGF receptors.

Topics: Cell Division; Cell Line; Cell Transformation, Viral; Culture Media; Epidermal Growth Factor; ErbB Receptors; Simian virus 40; Tretinoin

Murine fibroblasts transformed by the myc oncogene have a reduced growth factor dependence for both anchorage-dependent and anchorage-independent proliferation. Here we show that v-myc-transformed Balb/c 3T3 cells require, in addition to insulin, only platelet-derived growth factor (PDGF) or epidermal growth factor (EGF) for anchorage-dependent growth in serum-free media. PDGF, however, cannot efficiently be substituted by EGF for anchorage-independent growth. The results suggest that constitutive v-myc expression reduces cellular growth factor requirements by non-autocrine mechanisms for proliferation in monolayer cultures. In contrast, v-myc-transformed cells require plasma components and growth factors of the 'competence' type for anchorage-independent growth. We also demonstrate that the requirement for PDGF by the myc-transformed cells can be abrogated by v-K-ras, v-src and v-fos but not the v-raf oncogene. The results demonstrate that oncogenes can cooperate in the expression of the transformed phenotype by also drastically reducing cellular growth factor requirements.

Topics: Animals; Cell Division; Cell Transformation, Viral; Epidermal Growth Factor; Growth Substances; Insulin; Mice; Oncogenes; Platelet-Derived Growth Factor

Characterisation and quantitation of epidermal growth factor receptors (EGFR) have been carried out on eight human squamous carcinoma cell lines and the results compared with those from simian virus transformed keratinocytes and normal keratinocytes grown under similar conditions. All cells tested possess both high and low affinity receptors with dissociation constants ranging from 2.4 X 10(-10) M to 5.4 X 10(-9) M. When epidermal growth factor (EGF) binds to its receptor it is internalised and degraded and the receptor is down regulated. Malignant cells and virally transformed cells possess 5-50 times more EGF receptors than normal keratinocytes and one cell line LICR-LON-HN-5 possesses up to 1.4 X 10(7) receptors per cell, which is the highest number yet reported for a cell line. These results are discussed in the context of recent data that suggest that the increased expression of EGF receptors in epidermoid malignancies may be an important component of the malignant phenotype in these tumours.

Topics: Carcinoma, Squamous Cell; Cell Line; Cell Transformation, Viral; Chloroquine; Chromatography, High Pressure Liquid; Epidermal Cells; Epidermal Growth Factor; ErbB Receptors; Humans; Keratins; Receptors, Cell Surface; Time Factors

We have shown that MT depolymerization by colchicine and other drugs is sufficient to initiate DNA synthesis in serum-free cultures of embryonic fibroblasts and that stabilization of MTs with taxol inhibits this initiation. Growth factors and oncogenic DNA viruses also initiate DNA synthesis by a taxol-sensitive mechanism that appears to require MT depolymerization or rearrangements. Because we have shown that microtubule heterogeneity exists within single fibroblastic cells, we have carried out a series of experiments to determine the extent of microtubule disruption necessary to initiate DNA synthesis. We have compared the effects of various concentrations of colchicine and taxol on initiation of DNA synthesis with their effects on cytoplasmic MT complexes as visualized by indirect immunofluorescence microscopy and quantitated by direct binding of radiolabeled monoclonal antibody to cytoskeletons. The opposing effects of these drugs on MTs shows that there is a correlation between the extent of MT depolymerization and initiation of DNA synthesis. Initiation of DNA synthesis by colchicine in the presence of taxol is half-maximal when taxol and colchicine are added to cultures at a ratio of about 13 to 1. At this drug ratio, taxol stabilizes MTs near the nucleus, but MTs near the cell periphery are depolymerized. Maximal inhibition of DNA synthesis by taxol occurs only at taxol to colchicine ratios where MTs extend throughout the cytoplasm to the cell periphery. Thus, depolymerization of a small fraction of total MTs, particularly those near the periphery, may be sufficient to initiate proliferative events.

Topics: Alkaloids; Animals; Antineoplastic Agents; Cell Transformation, Viral; Cells, Cultured; Colchicine; Cytomegalovirus; DNA Replication; Embryo, Mammalian; Epidermal Growth Factor; Homeostasis; Mice; Microtubules; Paclitaxel; Thrombin

We have characterized two monoclonal antibodies which recognize human p36. These have been used to examine the sites and extent of serine and tyrosine phosphorylation of p36 in human cells treated with epidermal growth factor and platelet-derived growth factor and in human cells transformed with viruses whose oncogenes encode protein-tyrosine kinases.

Topics: Annexins; Antibodies, Monoclonal; Cell Transformation, Viral; Epidermal Growth Factor; Humans; Membrane Proteins; Oncogene Protein pp60(v-src); Oncogenes; Phosphorylation; Platelet-Derived Growth Factor; Protein-Tyrosine Kinases; Retroviridae Proteins; Serine; Tyrosine

Simian sarcoma virus, an acutely transforming primate retrovirus with capacity to induce gliomas and sarcomas in experimental animals, has acquired its transforming properties by transducing the cellular gene sequences that encode one of the constituent chains of platelet-derived growth factor. Suramin, a drug used in the treatment of trypanosomiasis and onchocerciasis, has previously been reported to inhibit the interaction of platelet-derived growth factor with its cell surface receptor. We show here that suramin efficiently reverts the simian sarcoma virus-induced transformed phenotype in human and rat fibroblasts and propose that this is due to neutralization of an externalized v-sis product. Moreover, we show that suramin inhibits the action of a broad spectrum of growth factors.

Topics: Animals; Avian Sarcoma Viruses; Calcium; Cell Cycle; Cell Transformation, Viral; Epidermal Growth Factor; ErbB Receptors; Humans; Mice; Platelet-Derived Growth Factor; Receptors, Cell Surface; Receptors, Platelet-Derived Growth Factor; Sarcoma Virus, Woolly Monkey; Sarcoma Viruses, Murine; Suramin

Transformation of an established Fischer rat embryo (CREF) cell line by bovine papillomavirus type 1 (BPV-1), in contrast to transformation by type 5 adenovirus or the T24 (Ha-ras) oncogene, resulted in transformants which did not exhibit a major increase in saturation density or a decrease in 125I-epidermal growth factor binding. BPV-1-transformed CREF clones did, however, grow in agar suspension culture and were tumorigenic in both nude mice and Fischer rats. The majority of transformed clones contained multiple extrachromosomal copies of BPV DNA. One transformed CREF clone contained integrated BPV-1 DNA which underwent sequence rearrangements following tumor formation in a Fischer rat and reestablishment in cell culture. BPV copy number varied in subclones of transformants isolated from the same monolayer focus, in agar-derived subclones of the same transformed focus, in tumors and in tumor-derived BPV-transformed CREF subclones. The degree of expression of specific transformation-related phenotypes, i.e., saturation density, growth in agar, and tumorigenicity, was not correlated with BPV copy number in transformed clones. Analysis of the biological properties of tumor-derived BPV-1-transformed CREF subclones indicated that certain transformants developed a stable increase in expression of transformation-related properties, a process termed "progression." TPA did not enhance the frequency of BPV-1 transformation or BPV DNA copy number in transformed CREF cells. The present study demonstrates that the CREF- transformation system can be utilized to study the molecular basis of BPV-1 transformation and should prove useful in studying the role of specific BPV-1 transforming proteins in regulating expression of the transformed phenotype and the mechanism by which transformed cells undergo progression of the transformed state.

Topics: Adenoviruses, Human; Animals; Bovine papillomavirus 1; Cell Cycle; Cell Line; Cell Transformation, Viral; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation; Mice; Neoplasms, Experimental; Papillomaviridae; Rats; Receptors, Cell Surface; Tetradecanoylphorbol Acetate; Virus Replication

Two methods have been used to examine epidermal growth factor (EGF) receptor distribution in human scalp and foreskin. The first employed [125I]EGF viable explants and autoradiography to determine the EGF binding pattern while the second used a monoclonal antibody to the human EGF receptor to map the distribution on frozen skin sections of an extracellular epitope on the EGF receptor. The [125I]EGF binding experiments showed accessible, unoccupied EGF receptors to be present on the epidermal basal cells (with reduced binding to spinous cells), the basal cells of the hair shaft and sebaceous gland, the eccrine sweat glands, capillary system, and the hair follicle outer root sheath, generally similar in pattern to that previously reported for full-thickness rat skin and human epidermis. The same areas also bound EGF-R1 but in addition the monoclonal antibody recognized a cone of melanin containing presumptive cortex cells, excluding the medulla, lying around and above the upper dermal papilla of anagen hair follicles, epithelial cells around the lower dermal papilla region, and in some tissue samples the cell margins of the viable differentiating layers of the epidermis. In a control study, to clarify whether EGF-R1 could recognize molecules unrelated to the EGF receptor, the EGF binding and EGF-R1 recognition profiles were compared on cultures of SVK14 cells, a SV40 transformed human keratinocyte cell line. EGF binding and EGF-R1 monoclonal antibody distribution on these cells was found to be similar, indicating that, at least for SVK14 cells, EGF-R1 binding provides a reliable marker for EGF binding. Explanations for the discrepancies between these two methods for determining EGF receptor distribution in human skin are discussed, including the possibility that latent EGF receptors, unable to bind [125I]EGF, may be present in some differentiating epithelial compartments.

Topics: Antibodies, Monoclonal; Binding Sites; Cell Transformation, Viral; Epidermal Growth Factor; ErbB Receptors; Fluorescent Antibody Technique; Humans; Iodine Radioisotopes; Receptors, Cell Surface; Simian virus 40; Skin; Staining and Labeling

Transformation of normal keratinocytes by simian virus 40 (SV 40) leads to the establishment of epithelial cell lines that can be cultured in the absence of the feeder layer and do not become senescent in culture. The SVK14 cell line developed by Taylor-Papadimitriou et al can serve as a model for study of the modification of various cellular processes by certain pharmacologic and physiologic agents, because these cells resemble normal keratinocytes with respect to a variety of parameters related to proliferation and differentiation, as follows: The SVK14 cells show the same ability to form ionophore-induced cross-linked envelopes that is strongly suppressed when the calcium level in the culture medium is reduced. When cultured in a high-calcium medium, both cell types showed a high rate of de novo cholesterol synthesis that was independent of the extracellular lipoprotein concentration. Cells cultured in a low-calcium medium had a much lower rate of cholesterol synthesis, but this rate increased markedly in cells preincubated in lipoprotein-deficient (LPDS) medium and decreased again with the addition of increasing amounts of low-density lipoprotein (LDL). Both types of cell showed decreased ability to bind epidermal growth factor (EGF) and LDL during calcium-induced differentiation, the expression of LDL and/or EGF receptors being high in low-calcium and low in high-calcium cells. Addition of etretinate (0.05-5.0 microM) suppressed cholesterol synthesis and strongly stimulated triglyceride synthesis in both cell types without significantly affecting the rate of protein synthesis. The addition of small doses of glucocorticoids (10(-9) to 10(-6) M) led to stimulation and higher doses (up to 5 X 10(-5) M) to inhibition of cell proliferation.

Topics: Calcium; Cell Differentiation; Cell Division; Cell Transformation, Viral; Cells, Cultured; Cholesterol; Epidermal Cells; Epidermal Growth Factor; Humans; Keratins; Lipid Metabolism; Lipoproteins, LDL; Male; Protein Biosynthesis; Receptors, Glucocorticoid; Simian virus 40

BALB-/MK-2 mouse epidermal keratinocytes required epidermal growth factor for proliferation and terminally differentiated in response to high Ca2+ concentration. Infection with retroviruses containing transforming genes of the src and ras oncogene families led to rapid loss of epidermal growth factor dependence, in some cases, accompanied by alterations in cellular morphology. The virus-altered cells continued to proliferate in the presence of high levels of extracellular calcium but exhibited alterations in normal keratinocyte terminal differentiation that appear to be specific to the particular oncogene. These alterations bore similarities to abnormalities in differentiation observed in naturally occurring squamous epithelial malignancies.

Topics: Animals; Calcium; Cell Differentiation; Cell Division; Cell Transformation, Viral; Cells, Cultured; Epidermal Cells; Epidermal Growth Factor; Epidermis; Keratins; Mice; Oncogenes; Protein-Tyrosine Kinases; Retroviridae

Epidermal growth factor (EGF) and an EGF-like transforming growth factor (eTGF) from retrovirally transformed cells bind to a common receptor type in A431 cells. We have investigated the effects of the tumor promoter phorbol myristate acetate [PMA] on EGF/eTGF receptors in intact A431 cells. Treatment with PMA at 37 degrees C induces a complete loss of high-affinity (Kd = 35-50 pM) binding sites for eTGF and EGF on the cell surface of A431 cells. This effect is half-maximal at 0.1 nM PMA, exhibits rapid kinetics, and persists for at least 4 hr in the presence of PMA. eTGF and PMA added to intact A431 cells induce the phosphorylation of immunoprecipitable 170kd EGF/eTGF receptors. The EGF/eTGF receptor isolated from control cells was found to contain phosphoserine and phosphothreonine. PMA and eTGF caused a marked increase in the level of these two phosphoamino acids. In addition, eTGF but not PMA caused the appearance of phosphotyrosine in the EGF/eTGF receptor in vivo. We conclude that the tumor-promoting phorbol diester regulates both the affinity and phosphorylation state of the A431 cell receptor for the type alpha transforming growth factors, eTGF and EGF.

Topics: Animals; Cell Line; Cell Transformation, Viral; Epidermal Growth Factor; ErbB Receptors; Peptides; Phorbols; Rats; Receptors, Cell Surface; Sarcoma Viruses, Feline; Tetradecanoylphorbol Acetate; Transforming Growth Factors

Chicken embryo fibroblasts sensitized by ts RSV respond to TGFs present in the media of non-transformed FR3T3 and NRK-4 rat cells and of the same cells transformed by KiMSV or RSV. They also respond to TGFs present in the media of BHK hamster cells transformed by MoMSV, PyV or RSV. Two other indicator rat cell lines, untransformed NRK-4 and FR3T3, sensitized by ts KiMSV, respond to the same TGF-containing media, and this response is increased by exogenous EGF. Normal FR3T3 cells failed to respond to any of the media. The most sensitive target cells were the ts KiMSV-FR3T3 cells at the restrictive temperature (39.5 degrees C). All the media tested on NRK-49F target cells required EGF for their TGF activity which was essentially dependent on prior activation by acidification. These data show that the above media from non-transformed or transformed cells contain beta-TGFs, with no detectable accompanying alpha-TGF activity. The release of and the response to these TGFs are not interdependent. A function of ts mutant src and k-ras viral oncogenes, still expressed at the restrictive temperature, can sensitize non-responsive cells, without there being any specificity towards the TGF producer cells.

Topics: Animals; Avian Sarcoma Viruses; Cell Line; Cell Transformation, Viral; Chick Embryo; Cricetinae; Culture Media; Epidermal Growth Factor; ErbB Receptors; Fibroblasts; Genes, Viral; Neoplasm Proteins; Peptides; Rats; Receptors, Cell Surface; Temperature; Transforming Growth Factors

An alpha-type transforming growth factor (TGF alpha) is produced at high levels by rat embryo cells transformed by the Snyder-Theilen strain of feline sarcoma virus (FeSV). Addition of 2 ng mouse epidermal growth factor (mEGF) during purification identified the presence of a second, EGF-dependent growth factor of the TGF beta type (TGF beta) in this conditioned medium. This factor had an approximate Mr of 12,000 and eluted at 37% acetonitrile during high performance liquid chromatography. This extracellular type of TGF beta activity also was present in conditioned medium of rat cells after infection with a transformation defective strain of Abelson leukemia virus, and hence expression of this growth factor activity was independent of cell transformation. Moreover, the presence of an EGF-dependent, 12,000 Mr clonogenic activity in extracts of bovine serum alone suggests serum as an origin for the B-type transforming growth factor initially observed in conditioned medium of Snyder-Theilen FeSV transformed cells. This does not, however, preclude the possibility that TGF beta is also secreted by the transformed rat embryo cells themselves.

Topics: Animals; Blood; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Chromatography, High Pressure Liquid; Culture Media; Embryo, Mammalian; Epidermal Growth Factor; Fibroblasts; Molecular Weight; Peptides; Rats; Sarcoma Viruses, Feline; Transforming Growth Factors

Topics: Amino Acid Sequence; Animals; Cell Transformation, Neoplastic; Cell Transformation, Viral; Epidermal Growth Factor; ErbB Receptors; Growth Substances; Humans; Oncogenes; Platelet-Derived Growth Factor; Receptors, Cell Surface

Recent advances in protein chemistry and genetic engineering have revealed new information about the molecular lesions involved in the induction and maintenance of cancer cells. It is now known that a single base change in the DNA of human cells leads to cancer. The normal pathway of proliferation and differentiation is perturbed by changes to molecules involved in the intracellular biochemical pathways controlled by growth factors. Some cancer cells appear to produce their own growth factor, others have higher concentrations of growth factor receptors on their surface and others have mutated versions of the intracellular proteins linked to the growth factor receptors. This increased understanding of growth control in normal and neoplastic cell populations is gradually providing a foundation for new approaches to cancer therapy.

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; DNA, Neoplasm; Epidermal Growth Factor; Growth Substances; Hematopoietic Stem Cells; Humans; Neoplasms; Neoplastic Stem Cells; Nerve Growth Factors; Platelet-Derived Growth Factor; Receptors, Cell Surface

Many growth factor receptors and retroviral transforming proteins share the property of phosphorylating proteins on tyrosine. Several substrates for both types of protein-tyrosine kinase have been identified. Treatment of quiescent cells with growth factors such as EGF and PDGF, whose receptors have ligand-stimulated protein-tyrosine kinase activities, induces tyrosine phosphorylation of three proteins, p45, p42 and p41. Two phosphorylated forms of p42 are found, the more basic of which is present in some but not all cells transformed by viral protein-tyrosine kinases. p42 is rapidly (as early as 1 min) but transiently (decreased to baseline by 2h) phosphorylated following PGDF or EGF treatment of quiescent fibroblasts. At saturating levels of mitogen the stoichiometry of p42 phosphorylation is greater than 50%. p42 is a highly conserved, rare (0.002% of total cell protein), soluble cytoplasmic protein. IGF I and insulin, whose receptors also have ligand-stimulated protein-tyrosine kinase activity, induce p42 phosphorylation in appropriate cells. In the case of insulin this effect has been observed in cells with large numbers of insulin receptors. p42 is also phosphorylated in response to mitogens whose receptors lack protein-tyrosine kinase activity, for example 12-O-tetradecanoylphorbol-13-acetate (TPA) and thrombin. For TPA there is evidence that this is an indirect effect due to the activation of a protein-serine/threonine kinase. On the basis of the highly conserved nature of this response and its generality, it seems likely that tyrosine phosphorylation of p42 is important for at least early responses to mitogens.

Topics: Animals; Cell Transformation, Viral; Epidermal Growth Factor; ErbB Receptors; Growth Substances; Humans; Insulin-Like Growth Factor I; Mitogens; Oncogene Proteins, Viral; Phosphorylation; Platelet-Derived Growth Factor; Protein Kinase C; Receptors, Cell Surface; Receptors, Platelet-Derived Growth Factor; Receptors, Somatomedin; Retroviridae; Tyrosine

NIH3T3 cells grow in a serum-free basal nutrient medium supplemented with fibronectin, transferrin, insulin, epidermal growth factor (EGF) and high density lipoprotein (HDL). The individual omission from the serum-free medium of insulin, EGF, or HDL results in greatly reduced cell growth. These growth-restrictive conditions can be used to select for cells transformed with SV40, the polyomavirus middle T antigen gene, the activated human ras gene, and the mouse c-myc gene.

Topics: Animals; Antigens, Polyomavirus Transforming; Antigens, Viral, Tumor; Blood; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Culture Media; Epidermal Growth Factor; Fibronectins; Insulin; Lipoproteins, HDL; Mice; Mice, Inbred C3H; Oncogene Proteins, Viral; Oncogenes; Simian virus 40; Transferrin

Type beta transforming growth factor (TGF-beta) is a two-chain polypeptide of 25,000 daltons isolated from many tissues, including bovine kidney, human placenta, and human platelets. It has been characterized by its ability to stimulate reversible transformation of nonneoplastic murine fibroblasts, as measured by the formation of colonies of these cells in soft agar (ED50 = 4 pM TGF-beta for NRK fibroblasts). We now show that the response of cells to TGF-beta is bifunctional, in that TGF-beta inhibits the anchorage-dependent growth of NRK fibroblasts and of human tumor cells by increasing cell cycle time. Moreover, the anchorage-independent growth of many human melanoma, lung carcinoma, and breast carcinoma cell lines is inhibited by TGF-beta at concentrations in the same range as those that stimulate colony formation of NRK fibroblasts (average ED50 = 10-30 pM TGF-beta for inhibition). Whereas epidermal growth factor and TGF-beta synergize to induce anchorage-independent growth of NRK fibroblasts, their effects on the growth of A-549 human lung carcinoma cells are antagonistic. The bifunctional response of cells to TGF-beta is further demonstrated in Fischer rat 3T3 fibroblasts transfected with a cellular myc gene. In these cells TGF-beta synergizes with platelet-derived growth factor to stimulate colony formation but inhibits the colony formation induced by epidermal growth factor. The data indicate that the effects of TGF-beta on cells are not a function of the peptide itself, but rather of the total set of growth factors and their receptors that is operant in the cell at a given time.

Topics: Animals; Cell Adhesion; Cell Cycle; Cell Line; Cell Transformation, Viral; Cells, Cultured; Drug Interactions; Epidermal Growth Factor; Fibroblasts; Growth Inhibitors; Growth Substances; Oncogenes; Peptides; Rats; Transforming Growth Factors

The recent discovery, that a vaccinia virus (VV) gene encodes a polypeptide with structural homology to transforming growth factor (TGF-alpha) and epidermal growth factor (EGF), led us to look for a virus-induced protein with the predicted biological activity. The supernatants of VV-infected cell cultures were found to contain an acid stable Mr 25,000 polypeptide that competes with EGF for binding to EGF membrane receptors. This VV-induced growth factor (VGF) like EGF and TGF-alpha is mitogenic and stimulates anchorage-independent cell growth in the presence of TGF-beta. However, VGF did not cross-react in a radioimmunoassay specific for small and large forms of TGF-alpha and exhibited minimal cross-reactivity with antisera to EGF. VGF was detectable in the culture medium within 2 hr, and maximal amounts were present 12 hr after infection. The level of VGF was proportional to the multiplicity of VV used. Inhibition of viral DNA synthesis enhanced VGF production, consistent with the hypothesis that VGF is an early gene product encoded by VV. The demonstration of a novel growth factor, released from cells infected with VV, may have important implications regarding the nature of virus-host interactions.

Topics: Animals; Cell Line; Cell Transformation, Viral; Cells, Cultured; Cercopithecus; Cross Reactions; DNA Replication; Epidermal Growth Factor; Fibroblasts; Growth Substances; Humans; Immune Sera; Infant, Newborn; Kidney; Male; Peptides; Skin; Transforming Growth Factors; Vaccinia virus

We have analyzed the effect of triiodothyronine and the tumor promoter 12-O-tetradecanoylphorbol-13-acetate on expression of the transformed phenotype, as monitored by agar clonigenicity, in a single cell clone of type 5 adenovirus-transformed Sprague-Dawley rat embryo cells, E11, and a nude mouse tumor-derived E11 subclone, E11-NMT. When grown in medium devoid of thyroid hormone, the cloning efficiency of E11 and E11-NMT cells was 1 and 19%, respectively, whereas continuous exposure to triiodothyronine (1 nm) resulted in cloning efficiencies of 3% for E11 cells and 50% for E11-NMT cells. In addition, the average size of agar colonies was larger when cells were grown in the presence of thyroid hormone. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (100 ng/ml) could substitute for triiodothyronine, resulting in agar cloning efficiencies similar to those found in cells grown in medium containing this hormone. In contrast, the presence or absence of triiodothyronine did not alter the population doubling time or saturation density of E11 or E11-NMT cells. Although both normal Sprague-Dawley rat embryo and type 5 adenovirus-transformed E11 and E11-NMT cells contained nuclear triiodothyronine receptors with similar affinities, the number of thyroid hormone receptors was lower in the virally transformed cells. Growth of E11-NMT cells in medium containing or lacking thyroid hormone did not alter the ability of these cells to bind 125I-labeled epidermal growth factor. In addition, the quantity of transforming polypeptide growth factor(s), secreted into serum-free medium, which was capable of competing with labeled epidermal growth factor for binding to its receptors on a cloned Fischer rat embryo cell line (CREF) and inducing agar growth in normally anchorage-dependent CREF cells was similar in E11-NMT cells grown in medium containing or depleted of thyroid hormone. Growth of E11-NMT cells in medium lacking triiodothyronine did not alter transcriptional rates of RNA produced by the viral transforming genes, E1a and E1b, or the level of a Mr 21,000 polypeptide encoded by E1b. These findings indicate that thyroid hormone can modulate expression of the transformed phenotype in type 5 adenovirus-transformed cells without directly modifying expression of type 5 adenovirus-transforming genes.

Topics: Adenoviruses, Human; Animals; Cell Adhesion; Cell Division; Cell Nucleus; Cell Transformation, Viral; Epidermal Growth Factor; Gene Expression Regulation; Peptide Biosynthesis; Rats; RNA, Messenger; Tetradecanoylphorbol Acetate; Transcription, Genetic; Transforming Growth Factors; Triiodothyronine

Chicken heart mesenchymal cells do not proliferate in culture medium containing heat-defibrinogenated plasma but proliferate briskly when incubated with epidermal growth factor (EGF) or brain fibroblast growth factor (bFGF) plus insulin-like growth factors (IGFs) or when infected with sarcoma or erythroblastosis viruses. When infected with the retrovirus MC29, which bears a v-myc oncogene, chicken heart mesenchymal cells proliferate at a more modest rate and become morphologically transformed. Heparin at 25 microgram/ml causes these MC29-transformed cells to become proliferatively quiescent and to assume a normal morphology. Heparin-treated MC29-infected cells are, however, 100 times more sensitive to EGF than are their normal, uninfected counterparts. MC29-infected cells appear, likewise, to be hypersensitive to bFGF and to PDGF preparations but not to insulin. We hypothesize, therefore, (i) that heparin prevents the generation by cells of a mitogen from plasma protein precursors in the culture medium; (ii) that the v-myc oncogene renders cells hypersensitive to EGF, bFGF, PDGF, and the putative plasma-protein-derived mitogen; and (iii) that MC29-infected cells must proliferate in order to manifest the transformed morphology. Chicken heart mesenchymal cells infected with a recombinant spleen necrosis virus containing a v-ras oncogene are morphologically transformed but proliferate only sluggishly in plasma-containing medium without added mitogenic hormones. Heparin does not significantly affect their behavior. They are refractory to mitogenic stimulation by EGF or bFGF suggesting that ras proteins mediate the effects of receptors for these hormones. The SNV/v-ras-infected cells proliferate briskly, however, in response to hyperphysiological concentrations of insulin, an IGF surrogate, and are considerably more sensitive to this IGF mitogenicity than are their normal, uninfected counterparts.

Topics: Animals; Cell Division; Cell Transformation, Viral; Chickens; Drug Synergism; Epidermal Growth Factor; Fibroblast Growth Factors; Heart; Heparin; Hot Temperature; Insulin; Myocardium; Oncogenes; Somatomedins

Serum stimulation of serum-deprived or density-inhibited normal cells enhances the level of various nutrient and ionic transport systems. Certain of these systems have been implicated in the regulation of cell proliferation. However, the use of serum stimulation to activate quiescent cells leads to enhancement of numerous transport systems with little understanding of which component or components of serum are related to activation of which transport systems. In this study we attempt to identify the specific effect of three known growth promoting factors (insulin, dexamethasone and epidermal growth factor [EGF]) on the activation of four membrane transport systems (A-amino acids, L-amino acids, glucose and K+) in normal and SV40-transformed WI38 human fibroblasts. We have also evaluated the effect of these growth factors on the stimulation of DNA synthesis in growth factor deprived cells. Thus, we can correlate the effect on a given transport system with the relative mitogenic stimulation produced by the growth factor. We conclude a) that a growth factor can effect a transport system differently in a normal versus transformed cell, b) that a specific growth factor can effect multiple transport systems and, c) with the exception of K+ transport, enhanced transport induced by a given growth factor does not necessarily correlate with the mitogenic potency of the growth factor. This latter point is of particular significance since the activation of K+ transport reflects, based on other studies, activation of the Na+-H+ exchanger which has been implicated in cell-cycle activation.

Topics: Amino Acids; Biological Transport, Active; Cell Division; Cell Membrane Permeability; Cell Transformation, Viral; Cells, Cultured; Culture Media; Dexamethasone; DNA; Embryo, Mammalian; Epidermal Growth Factor; Fibroblasts; Glucose; Growth Substances; Humans; Insulin; Lung

Normal chicken heart mesenchymal cells at low density in monolayer culture in plasma-containing medium have a polygonal shape and are proliferatively quiescent. The combination of epidermal growth factor and insulin at hyperphysiological concentration, an insulin-like growth factor surrogate, causes these cells to assume a fusiform shape and to increase 40-fold in number during four days of incubation. These mitogenic hormones do not, however, induce normal chicken heart mesenchymal cells to form colonies in agarose suspension culture. Chicken heart mesenchymal cells infected with the Schmidt-Ruppin or Prague-A strains of Rous sarcoma virus or with the Fujinami or Y73 avian sarcoma viruses assume spindle and round shapes, increase 50-100 fold in number during four days of monolayer culture in the absence of mitogenic hormones and form macroscopic colonies during 3-4 days of agarose suspension culture. The autonomous (mitogenic hormone-independent) proliferation, in monolayer culture, of cells infected with temperature-sensitive transformation mutants of Rous sarcoma virus (tsNY68, tsNY72, tsLA24, tsLA29) is temperature-sensitive. Chicken heart mesenchymal cells infected with avian erythroblastosis virus assume spindle shapes and proliferate in monolayer culture at a rate comparable to that of sarcoma virus-infected cells but do not, however, form colonies in agarose suspension culture. Cells infected with the myelocytomatosis virus MC29 assume stellate shapes and increase 18-fold in number during four days of monolayer culture. Cells infected with the myelocytomatosis virus MH2 assume fusiform shapes and increase fourfold in number during four days of monolayer culture. Neither MC29 nor MH2 renders chicken heart mesenchymal cells capable of colony formation in agarose suspension culture. Infection with avian leukosis viruses (RAV-1, RAV-2, RPL-42) or with transformation-defective mutants of Rous sarcoma virus (tdNY105, 107, 109) does not affect the morphology or proliferative behavior of chicken heart mesenchymal cells. Monolayer culture of chicken heart mesenchymal cells in plasma-containing medium appears, therefore, to define the ability of onc genes of acute transforming avian retroviruses to induce autonomous (mitogenic hormone-independent) cell proliferation, the essential characteristic of neoplasia. The differences in transformed morphology and rates of autonomous proliferation between cells infected with different acute transforming retroviruses

Topics: Alpharetrovirus; Animals; Avian Leukosis Virus; Avian Myeloblastosis Virus; Avian Sarcoma Viruses; Cell Division; Cell Transformation, Viral; Cells, Cultured; Chickens; Colony-Forming Units Assay; Epidermal Growth Factor; Insulin; Myocardium; Temperature

Two flat cellular revertant cell lines, F-2 and C-11, which were originally selected from the DT line of Kirsten murine sarcoma virus (Ki-MuSV)-transformed NIH/3T3 cells, were examined for the production of transforming growth factors (TGFs). The revertant cells fail to grow in semisolid medium as colonies and exhibit a markedly reduced level of tumorigenicity in nude mice, although they are known to express high levels of p21ras, the product of the Kirsten sarcoma virus oncogene, ras, and they contain a rescuable transforming virus. TGF activity associated with the transformed, revertant, and non-transformed cell lines was measured by the ability of concentrated conditioned medium (CM) from these cells to induce normal rat kidney (NRK) and NIH/3T3 cells to form colonies in semisolid agar suspension cultures and to inhibit the binding of 125I epidermal growth factor (EGF) to specific cell surface receptors. CM from the transformed DT cells and from both the F-2 and C-11 revertants contains TGF activity, in contrast to CM obtained from normal NIH/3T3 cells. Furthermore, unlike NIH/3T3 cells, neither the DT nor the revertant cells were able to bind 125I EGF. All four cell lines were able to proliferate in serum-free medium supplemented with transferrin, insulin, EGF, and Pedersen fetuin. However, in basal medium lacking these growth factors, only DT cells and, to a lesser extent, the revertant cells were able to grow. These results suggest that the F-2 and C-11 revertants fail to exhibit all of the properties associated with transformation because the series of events leading to the transformed phenotype is blocked at a point(s) distal both to the expression of the p21 ras gene product and also to the production of TGFs and that the production of TGFs may be necessary but not sufficient for maintaining the transformed state.

Topics: Animals; Cell Adhesion; Cell Division; Cell Transformation, Viral; Epidermal Growth Factor; ErbB Receptors; Growth Substances; Kirsten murine sarcoma virus; Mice; Oncogenes; Peptide Biosynthesis; Receptors, Cell Surface; Sarcoma Viruses, Murine; Sarcoma, Experimental; Transforming Growth Factors

We have measured the production of a basic-somatomedin-like activity (SLA) by a variety of human tumor-derived, transformed, and normal postnatal cell cultures; and we have compared the production of SLA by these cell types with the production of SLA by adult rat hepatocytes cultured in serum-free medium. Cells derived from a human epidermoid carcinoma (KB), a pancreatic carcinoma (Panc-1), a Simian virus 40 transformed adult human skin-derived cell line (SV40 fibroblasts), and a normal adult human skin-derived fibroblast line released SLA when cultured in a serum-free growth medium. No SLA was recovered from the culture medium of human choriocarcinoma-derived cells (BeWo) or of a human lymphoblastoid cell line (IM-9). The production of SLA by rat hepatocytes cultured in serum-free medium appeared to exceed the production of SLA by the other cell cultures. In cultures of KB cells, SV40 fibroblasts, and rat hepatocytes, the production of SLA depended on the frequency with which the growth medium was renewed; in general, the highest rates of SLA production were observed when the medium was renewed every 48-72 h. The presence of mouse epidermal growth factor (urogastrone) (EGF-URO) in the serum-free culture medium stimulated the production of SLA by KB cells and by rat hepatocytes, but did not increase SLA production by normal or by SV40-transformed human skin-derived fibroblasts. We conclude that tumor-derived cells are capable of producing somatomedin-like activity and that the production of SLA by such cells can be subject to controls (nutrient availability, EGF-URO stimulation) that regulate SLA production, either by normal adult tissues, like liver, or by a variety of normal embryonic tissues.

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cells, Cultured; Epidermal Growth Factor; Humans; Liver; Mice; Neoplasms; Simian virus 40; Somatomedins

Topics: Animals; Annexins; Cell Transformation, Neoplastic; Cell Transformation, Viral; DNA; Epidermal Growth Factor; ErbB Receptors; Guanosine Triphosphate; Humans; Membrane Proteins; Neoplasm Proteins; Nucleoproteins; Oncogene Protein pp60(v-src); Oncogenes; Phosphotyrosine; Platelet-Derived Growth Factor; Protein Binding; Protein Biosynthesis; Protein Kinases; Receptors, Cell Surface; Retroviridae; Tyrosine; Viral Proteins

Four psychoactive agents which inhibit calmodulin activity were used to study their effect on the binding of epidermal growth factor (EGF) to normal and simian-virus-40-transformed human fibroblasts (WI38). These calmodulin antagonists decreased the binding of 125I-labelled EGF to the transformed, but not to the normal, cell in a dose-dependent manner. The mechanism of this effect appears to be due to a decrease in the apparent affinity of the plasma-membrane EGF receptor for the EGF molecule.

Topics: Calmodulin; Cell Transformation, Viral; Cells, Cultured; Chlorpromazine; Epidermal Growth Factor; Fibroblasts; Haloperidol; Humans; Imipramine; Simian virus 40; Trifluoperazine

When KB cells labeled with 51Cr (1 muCi/ml) were treated with adenovirus type 2 (Ad2) at pH 6, 51Cr was released in a concentration-dependent manner with half-maximal release at 1 microgram/ml of virus. The 51Cr release was maximum at 10 micrograms/ml and represented nearly 20% of cell-associated 51Cr. 51Cr release depended on the length of incubation with Ad2 and the pH of the medium; maximum release was at pH 6 with very little release at pH 7.5. An antiserum against the penton base of Ad2 specifically neutralized the ability of Ad2 to release 51Cr. Chloroquine up to 200 microM did not block Ad2-dependent 51Cr release. DEAE-dextran stimulated Ad2-dependent 51Cr release; 4-5-fold stimulation was observed at 50 micrograms/ml of DEAE-dextran. We have compared the ability of Ad2 to release 51Cr with its ability to disrupt endocytic vesicles. Vesicle disruption was independent of pH of the medium in the range of pH 6 to 7.5 and was blocked by chloroquine, whereas, 51Cr release was much greater at pH 6 than at pH 7.5 and was not affected by chloroquine. These results suggest that Ad2 possess a lytic activity which ordinarily lyses acidic endocytic vesicles but, if the cells are maintained at acidic pH, can also directly disrupt the plasma membrane.

Topics: Adenoviruses, Human; ADP Ribose Transferases; Bacterial Toxins; Carcinoma; Cell Transformation, Viral; Chloroquine; Chromium Radioisotopes; Epidermal Growth Factor; Exotoxins; Humans; Hydrogen-Ion Concentration; KB Cells; Kinetics; Mouth Neoplasms; Pseudomonas aeruginosa Exotoxin A; Virulence Factors

Topics: Animals; Cell Division; Cell Transformation, Viral; Epidermal Growth Factor; Molecular Weight; Muscle Proteins; Oncogene Protein pp60(v-src); Phosphoproteins; Platelet-Derived Growth Factor; Protein Kinases; Protein-Tyrosine Kinases; Vinculin; Viral Proteins

Topics: Avian Sarcoma Viruses; Cell Transformation, Viral; Epidermal Growth Factor; Escherichia coli; Gene Expression Regulation; Oncogene Protein pp60(v-src); Phosphoprotein Phosphatases; Phosphotyrosine; Protein Kinases; Tyrosine; Viral Proteins

Protein kinases that phosphorylate the hydroxyl group of tyrosine residues of proteins have been implicated in cell transformation by some retroviruses and in regulation of normal cell growth by some polypeptide growth factors. To facilitate the identification of tyrosine kinase substrates, we developed monoclonal antibodies to the hapten azobenzylphosphonate. One of these antibodies, MA-2G8, proved to be especially attractive in that it bound a derivative of aminophenylphosphate, a close phosphotyrosine analog, with higher affinity than it bound the corresponding derivative of aminobenzylphosphonate; however, its affinity for phosphoserine was negligible. In this paper we describe the optimal conditions for using this antibody to isolate phosphotyrosine proteins, emphasizing particularly that its interaction with phosphotyrosyl proteins is sensitive to ionic detergents and to antibody density on the immunosorbent matrix. The antibody also bound ATP citrate lyase; this enzyme lacks phosphotyrosine but contains phosphohistidine, which is similar structurally to phosphotyrosine. By attaching the antibody at high density to Sepharose beads and omitting ionic detergents from the buffers, it was possible by microbatch immunoadsorption (followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) to isolate the 120,000-dalton transforming protein and several other phosphotyrosyl proteins from cells transformed by Abelson murine leukemia virus. Under the same conditions, phosphotyrosyl proteins were also isolated from human epidermal carcinoma cells (A431) that had been stimulated with epidermal growth factor; most prominent among these proteins was the 170,000-dalton receptor for epidermal growth factor.

Topics: Animals; Antibodies, Monoclonal; Cell Division; Cell Transformation, Viral; Cells, Cultured; Epidermal Growth Factor; Humans; Mice; Phosphoproteins; Phosphotyrosine; Protein Kinases; Tyrosine

Clonal BALB/c mouse epidermal keratinocyte (BALB/MK) cell lines were established in tissue culture. Despite their aneuploid nature, the lines were nontumorigenic, and retained in vitro properties similar to those of primary diploid keratinocytes. These included the constitutive expression of keratin and terminal differentiation in response to a calcium concentration greater than 1.0 mM in the medium. The cells also demonstrated an absolute requirement for nanomolar concentrations of epidermal growth factor (EGF) for their proliferation. BALB or Kirsten murine sarcoma viruses are acute transforming retroviruses, which have been shown to transform fibroblastic and hematopoietic cells. Infection of BALB/MK or its clonal sublines with either virus leads to the rapid acquisition of EGF-independent growth. The cells concomitantly lose their sensitivity to calcium-induced terminal differentiation. Thus these retroviruses can rapidly confer upon epithelial keratinocytes in culture growth properties that resemble those of malignant epidermoid carcinoma cells.

Topics: Animals; Calcium; Cell Differentiation; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Clone Cells; Epidermal Cells; Epidermal Growth Factor; Kirsten murine sarcoma virus; Mice; Mice, Inbred BALB C; Sarcoma Viruses, Murine

Topics: Avian Sarcoma Viruses; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Cell Transformation, Viral; Epidermal Growth Factor; Glycopeptides; Humans; Membrane Proteins; Oncogene Protein pp60(v-src); Phosphorylation; Protein Kinases; Substrate Specificity; Viral Proteins

The proliferative response of nontransformed rat embryo (Rat-1) cells and avian sarcoma virus-transformed B31 cells to high-density lipoprotein (HDL), transferrin, insulin, epidermal growth factor (EGF), and fibroblast growth factor has been compared. HDL, added in combination with transferrin, supported the active proliferation of low-density cultures of both Rat-1 and B31 cells. No major difference in the sensitivity of Rat-1 or B31 cells to HDL and transferrin was observed when cells were maintained on dishes coated with an extracellular matrix (ECM) obtained from bovine corneal endothelial cells. The two cell types differed in their response to the other known growth-promoting agents, however, in contrast to Rat-1 cells, transformed B31 cells no longer respond to EGF and fibroblast growth factor and respond only inconsistently to the mitogenic stimulus of insulin. Nontransformed Rat-1 cells and transformed B31 cells grown in the presence of medium containing, respectively, HDL, transferrin, insulin, EGF, and dexamethasone or HDL, transferrin, and insulin could be subcultured for more than 50 generations in the complete absence of serum without significant alteration in morphology, growth rate, or tumorigenicity (B31 cells). When plastic or collagen-coated dishes were used as the substrate instead of ECM-coated dishes, nontransformed Rat-1 cells grew very slowly in the serum-free medium described above. Dishes coated with collagen were not more efficient than was plastic in supporting growth of Rat-1 cells under these conditions. Coating dishes with fibronectin, however, clearly improved their growth, bringing the final cell density of the cultures up to 50% of that obtained on ECM-coated dishes. In contrast, transformed B31 cells grew significantly in serum-free medium when seeded on plastic or collagen-coated dishes, and the final cell density reached by cells on these substrates was 50% of that of cells maintained on ECM-coated dishes. In addition, B31 cells grew equally well when seeded on fibronectin- or ECM-coated dishes. The transformed cells thus showed less stringent substrate requirements when grown under serum-free conditions than did nontransformed Rat-1 cells. Our data also indicate that HDL, in combination with transferrin, supported efficient anchorage-independent growth of B31 cells. Fibroblast growth factor, but not insulin or EGF, further improved anchorage-independent growth of these cells. The capacity of cells to form colonies in s

Topics: Animals; Avian Sarcoma Viruses; Cell Division; Cell Line; Cell Transformation, Viral; Culture Media; Dexamethasone; Dose-Response Relationship, Drug; Embryo, Mammalian; Epidermal Growth Factor; Growth Substances; Insulin; Lipoproteins, HDL; Rats; Transferrin; Tumor Virus Infections

Lowering of extracellular Ca2+ levels will reversibly arrest the growth of human fibroblasts (WI38). Simian virus40(SV40)-transformed WI38 cells do not exhibit this Ca2+-dependent arrest. One possibility for this difference in Ca2+ requirement is that extracellular or surface membrane-bound Ca2+ may be required for growth factor receptor-mediated endocytosis and this Ca2+ requirement may differ in normal versus transformed cells. In this study we have evaluated the role of Ca2+ in the binding, internalization, and degradation of epidermal growth factor (EGF) in the WI38 and SV40WI38 cell. The binding of [125I]EGF to the cell surface is not significantly altered by lowering of Ca2+ to 10(5)-M levels in either the normal or transformed cell. At this Ca2+ level, growth of the normal cell is inhibited. The subsequent internalization of EGF is reduced nearly threefold in the normal cell but not in the transformed cell following Ca2+ deprivation. Degradation of the EGF-receptor complex is also sensitive to Ca2+. A twofold reduction in the rate of release of acid-soluble 125I occurs in the normal but not the transformed cell under conditions of lowered medium Ca2+. In contrast, 2-chloro-10-3-aminopropyl phenothiazine (CP), an inhibitor of the Ca2+-dependent regulator protein calmodulin, causes an inhibition of [125I]EGF internalization and degradation in both the normal and transformed WI38 cell, and a marked inhibition of [125I]EGF binding to the cell surface receptor of the transformed cell but not the normal cell.

Topics: Calcium; Cell Transformation, Viral; Cells, Cultured; Endocytosis; Epidermal Growth Factor; ErbB Receptors; Humans; Phenothiazines; Protein Binding; Receptors, Cell Surface; Simian virus 40

Transforming growth factors (TGFs) are a heterogeneous family of polypeptides that induce anchorage-independent growth in nonneoplastic anchorage-dependent cells. They have been found in many tissues, both neoplastic and nonneoplastic. All TGFs isolated thus far are of low molecular weight (6000-25,000), are acid and heat stable, and are inactivated by reagents that reduce disulfide bonds. TGFs have been classified as type alpha or type beta based on their interactions with the receptor for epidermal growth factor (EGF) and their requirement for EGF (or an EGF-like polypeptide) for functional activity. TGF-alpha and TGF-beta act synergistically. TGF-alpha induces phosphorylation of tyrosine in the EGF receptor. TGF-beta, isolated from bovine sources, accelerates experimental wound healing in rats.

Topics: Animals; Cell Adhesion; Cell Division; Cell Transformation, Neoplastic; Cell Transformation, Viral; Drug Stability; Epidermal Growth Factor; ErbB Receptors; Humans; Mice; Molecular Weight; Neoplasms; Neoplasms, Experimental; Peptides; Rats; Receptors, Cell Surface; Transforming Growth Factors

In an attempt to analyse the cause-effect relationship between anchorage-independent growth (a property which correlates best with in vivo tumorigenicity) and a set of other common transformation-related properties, the effect of retinoic acid (RA) treatment on six unrelated transformed cell lines (including DNA tumor virus, retrovirus, and spontaneously transformed cells) were studied. The data show that the changes in morphology and cellular orientation in culture, loss of cell surface fibronectin, disruption of actin microfilaments, increased hexose uptake, loss of density-dependent growth, and decreased binding of EGF, properties which are often associated with oncogenic transformation of cells, are dissociable from one another and from anchorage-independent growth. RA appears to interfere with anchorage-independent growth of all the retrovirus and spontaneously transformed cell lines (responsive cells) that we examined; however, such treatment failed to inhibit anchorage-independent growth in both of the DNA tumor virus-transformed cell lines (non-responsive cells) that we used in the present study. The presence of RA-binding proteins in both responsive and non-responsive cells suggests that the mechanism of RA action for the inhibition of anchorage-independent growth in transformed cells may be independent of the presence of such cytoplasmic proteins. Finally, the present study clearly indicates that the use of RA treatment, like partial transformation mutants of oncogenic viruses, can be a novel approach in analysing the general mechanism by which transformed cells grow without anchorage.

Topics: Actins; Animals; Carrier Proteins; Cell Adhesion; Cell Transformation, Viral; Cytoplasm; Epidermal Growth Factor; Fibronectins; Glucose; Mice; Mice, Inbred BALB C; Receptors, Retinoic Acid; Tretinoin

The transforming proteins (p21) of Harvey and Kirsten sarcoma viruses threonine kinase activity, which phosphorylates threonine 59 of the p21 proteins themselves. A tridecapeptide: Arg-Arg-Leu56-Asp-Thr-Thr59-Gly-Gln-Glu-Tyr-Ser-Ala66 containing residues 56-66 of p21 is phosphorylated solely on tyrosine by the epidermal growth factor (EGF)-stimulated tyrosine kinase of A431 cell membranes. Km-Values of 240 and 80 microM and Vmax values of 1.7 and 0.1 nmol.min-1.mg-1 were obtained in the presence and absence of EGF, respectively.

Topics: Carcinoma, Squamous Cell; Cell Line; Cell Transformation, Viral; Epidermal Growth Factor; Humans; Kinetics; Kirsten murine sarcoma virus; Peptides; Phosphorylation; Protein Kinases; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Sarcoma Viruses, Murine; Transforming Growth Factors

Cloned cells of the rat kidney fibroblast line designated NRK-49F, which requires epidermal growth factor (EGF), fibronectin, insulin, and retinoic acid for rapid multiplication in serum-free culture, were transformed by polyoma virus. Cells from two independent transformation events were isolated and cloned, as were cells from two corresponding control untransformed cultures not treated with virus. Tests in serum-free culture showed that the two transformed subclones required EGF and fibronectin but not insulin or retinoic acid for rapid multiplication, whereas the two control subclones retained the requirements for all four factors. Although EGF at 10 to 50 ng/ml stimulated the multiplication of all four subclones, EGF at 500 ng/ml strongly inhibited multiplication of the two transformed subclones but not the two control subclones.

Topics: Animals; Cell Transformation, Viral; Clone Cells; Dose-Response Relationship, Drug; Epidermal Growth Factor; Fibronectins; Insulin; Kidney; Polyomavirus; Rats; Tretinoin

Sarcoma growth factor (SGF) derived from conditioned medium of Moloney sarcoma virus-transformed cells and partially purified by gel filtration (crude SGF) has been characterized by its ability both to compete with epidermal growth factor (EGF) for binding to membrane receptors and to induce anchorage-independent growth of untransformed cells. We now show that further purification of crude SGF by reverse-phase HPLC on muBondapak C18 and CN columns at pH 2 resolves it into two distinctly different polypeptides, which we call types alpha and beta transforming growth factors (TGFs). Type alpha TGF (TGF-alpha), but not type beta TGF (TGF-beta), competes for binding to the EGF receptor and induces the formation of small colonies (1,000-2,000 micron2) of normal rat kidney cells in soft agar. Both TGF-beta and EGF or TGF-alpha must be present in order to induce the formation of large colonies (7,000-15,000 micron2). Based on EGF competing equivalents as determined from a radioreceptor assay with 125I-labeled EGF in normal rat kidney cells, the relative ability of EGF and TGF-alpha to potentiate TGF-beta-dependent colony formation is in the order conditioned-medium TGF-alpha greater than EGF greater than intracellular TGF-alpha. Suboptimal concentrations of the same polypeptides give additive potentiation of the TGF-beta-dependent colony-forming response; saturating levels potentiate a similar maximum response whether used alone or in various combinations. The data indicate that the EGF-competing activity of crude SGF is due to its TGF-alpha component alone, whereas the soft-agar colony-forming activity is due to the combined action of two distinct polypeptides, TGF-alpha and TGF-beta.

Topics: Animals; Cell Division; Cell Transformation, Viral; Cells, Cultured; Epidermal Growth Factor; Growth Substances; Macromolecular Substances; Moloney murine leukemia virus; Peptides; Rats; Transforming Growth Factors

Topics: Animals; Blood; Cell Adhesion; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Clone Cells; Cricetinae; Culture Media; Epidermal Growth Factor; Fibroblast Growth Factors; Insulin; Kidney; Peptides

Dihydrocytochalasin B (H2CB) disrupts the actin structure of Swiss/3T3 mouse fibroblasts and inhibits the ability of serum growth factors to stimulate DNA synthesis in quiescent cultures. Low doses of H2CB (2-10 X 10(-7) M) added to serum-arrested cells reversibly block initiation of DNA synthesis by serum; by epidermal growth factor and insulin; or by epidermal growth factor, fibroblast growth factor and insulin. H2CB is effective only when added to cells within 8-10 hr after stimulation. Low doses of H2CB cause cell rounding and a loss of actin microfilament bundles, but they do not interfere with glucose or thymidine transport. These results suggest that stimulation of 3T3 cells involves at least one obligatory actin-mediated step. Transformed cells appear to obviate this step, for H2CB does not inhibit the entry into S phase of SV40-transformed or Moloney murine sarcoma virus-transformed 3T3 cells synchronized by mitotic shake-off.

Topics: Actins; Animals; Biological Transport; Blood; Cell Line; Cell Transformation, Viral; Cytochalasin B; Cytoskeleton; DNA; Dose-Response Relationship, Drug; Epidermal Growth Factor; Fibroblast Growth Factors; Glucose; Insulin; Macromolecular Substances; Mice; Peptides; Thymidine

Topics: Animals; Blood; Cell Adhesion; Cell Count; Cell Division; Cell Line; Cell Movement; Cell Survival; Cell Transformation, Viral; Culture Media; Epidermal Growth Factor; Fibroblast Growth Factors; Growth Substances; Mice; Peptides; Platelet-Derived Growth Factor

We have studied the effects of paraquat (methyl viologen), a herbicide that increases intracellular production of superoxide radical, on the viability of virus-transformed and nontransformed normal rat kidney (NRK) cells in culture. We have shown that a low concentration of paraquat (12.5 microM) is cytotoxic toward virus-transformed cell lines, including Kirsten sarcoma virus- and SV40-transformed NRK cells. The corresponding untransformed NRK cells were resistant to the same and a 4-fold higher concentration of paraquat. There was a good correlation between the susceptibility of transformed and untransformed cells to paraquat cytotoxicity and their ability to increase the superoxide dismutase (SOD) enzymatic activity. We found that paraquat is cytotoxic toward Kirsten sarcoma virus-transformed and SV40-transformed NRK cells which showed low intracellular SOD activity. The relationship between SOD activity and paraquat cytoxicity was strengthened by the finding that the tolerance of NRK cells to the drug was associated with high intracellular SOD activity. This report also describes the isolation of a revertant (revertant RE8G3) cell line derived from Kirsten sarcoma virus-transformed NRK cells after paraquat treatment which contains SOD activity at levels much higher than those found in NRK cells. This revertant is undistinguishable from NRK cells with respect to its lack of transformed cell properties. Not only are these cells normal morphologically but also they do not grow in soft agar, an in vitro property that closely correlates with in vivo tumorigenicity. Several biological and biochemical properties of RE8G3 cells, including growth characteristics, surface receptors for both transferrin and epidermal growth factor (EGF), and the EGF-dependent 32P phosphorylation of specific membrane polypeptides have been studied. The most interesting conclusion that can be drawn from these studies is that there is a correlation between loss of the transformed phenotype and an increase in both EGF receptors and EGF-dependent 32P phosphorylation of a m.w. 170,000 membrane-associated protein.

Topics: Animals; Cell Division; Cell Line; Cell Survival; Cell Transformation, Viral; Drug Resistance; Epidermal Growth Factor; Kidney; Kirsten murine sarcoma virus; Paraquat; Phenotype; Phosphorylation; Protein Binding; Rats; Simian virus 40; Superoxide Dismutase; Time Factors; Tumor Virus Infections

Transforming growth factors (TGF) are growth-promoting polypeptides that cause phenotypic transformation and anchorage-independent growth of normal cells. They have been isolated from several human and animal carcinoma and sarcoma cells. One TGF is sarcoma growth factor (SGF) which is released by murine sarcoma virus-transformed cells. Whereas the TGF interacts with epidermal growth factor (EGF) cell membrane receptors, it is not detectable in culture fluids from cells which contain high numbers of free EGF cell membrane receptors. The SGF acts as a tumor promoter in cell culture systems, and its effect on the transformed phenotype is blocked by retinoids (vitamin A and synthetic analogs). The production of TGF by transformed cells and the responses of normal cells to the addition of TGF to the culture medium raise the possibility that cells "autostimulate" their growth by releasing factors that rebind at the cell surface. The term "autocrine secretion" has been proposed for this type of situation in which a cell secretes a hormone-like substance for which it has external cell membrane receptors. The autocrine concept may provide a partial explanation for some aspects of tumor cell progression.

Topics: Animals; Binding Sites; Carcinogens; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cells, Cultured; Epidermal Growth Factor; ErbB Receptors; Growth Substances; Humans; In Vitro Techniques; Neoplasms; Peptides; Receptors, Cell Surface; Sarcoma Viruses, Murine; Transforming Growth Factors

Topics: Actins; Animals; Cell Adhesion; Cell Transformation, Viral; Cytoskeleton; Epidermal Growth Factor; Fibronectins; Glucose; Kirsten murine sarcoma virus; Mice; Rats; Sarcoma Viruses, Murine; Temperature; Viral Proteins

The down regulation of surface membrane receptors for 125I) epidermal growth factor (EGF) has been evaluated in normal and SV40-transformed human fibroblasts (WI38) under conditions of serum-supplemented versus defined growth media. Both normal and transformed WI38 cells down regulate and recover the EGF receptor and these processes do not differ significantly in serum-supplemented versus defined media. These data are in contrast to a recent study that reported that the HeLa cell does not down regulate the EGF receptor in defined media, whereas it does in serum-supplemented media.

Topics: Cell Division; Cell Line; Cell Transformation, Viral; Culture Media; Epidermal Growth Factor; ErbB Receptors; Humans; Receptors, Cell Surface; Simian virus 40

A chloromethyl ketone derivative of lactic acid was shown to inhibit protein phosphorylation in plasma membranes of Ehrlich ascites tumor cells [Johnson, H. J., Zimniak, A., & Racker, E. (1982) Biochemistry 21, 2984-2989]. We now show that this inhibitor as well as three halomethyl ketone derivatives of amino acids and peptides specifically inhibits tyrosine protein kinase activity in intact plasma membranes and Triton extracts of plasma membrane of A-431 tumor cells. The most effective inhibitor is a bromomethyl ketone derivative of leucine that inhibits the phosphorylation of a protein that migrates to the same position as the EGF receptor in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Inhibition of phosphorylation took place in the presence or absence of added EGF, and the inhibitor did not interfere with the binding of EGF to the receptor nor with the dephosphorylation of the EGF-stimulated phosphoprotein. EGF-dependent phosphorylation in a Triton extract of plasma membranes from normal placenta was considerably less sensitive to the bromomethyl ketone derivative of leucine. The tyrosine protein kinase activity of the transformation gene product of Fujinami virus was particularly sensitive to the bromomethyl ketone derivative of leucine, while the src gene product of Rous sarcoma virus was comparatively less sensitive. The bromomethyl ketone inhibitor interfered with the phosphorylation of the EGF receptor by [gamma-32P]-8-azido-ATP but much less with the light-sensitive binding. This observation and the lack of interference with EGF binding suggest that the inhibitor interacts with the protein kinase portion of the receptor complex.

Topics: Avian Sarcoma Viruses; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Cell Transformation, Viral; Epidermal Growth Factor; Humans; Ketones; Kinetics; Phosphorylation; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Retroviridae; Structure-Activity Relationship

Topics: Animals; Carboxy-Lyases; Cell Adhesion; Cell Division; Cell Transformation, Viral; Cytoskeleton; Epidermal Growth Factor; Fibronectins; Mice; Ornithine Decarboxylase; Tretinoin

The Gardner and Snyder-Theilen isolates of feline sarcoma virus (FeSV) have previously been shown to encode high-molecular-weight polyproteins with a transforming function and an associated tyrosine-specific protein kinase activity. Cells transformed by these viruses exhibited morphological alterations, elevated levels of phosphotyrosine, and a reduced capacity for binding epidermal growth factor. In addition, polyproteins encoded by both of these FeSV isolates bound to, and phosphorylated tyrosine acceptor sites within, a 150,000-molecular-weight cellular substrate (P150). McDonough FeSV-transformed cells resembled Gardner and Snyder-Theilen FeSV transformants with respect to morphological changes and a reduced capacity for epidermal growth factor binding. in contrast to the other two FeSV isolates, however, McDonough FeSV encoded as its major translational product a high-molecular-weight polyprotein with probable transforming function but without protein kinase activity detectable under similar assay conditions. Moreover, total cellular levels of phosphotyrosine remained unaltered in McDonough FeSV-transformed cells, and the major McDonough FeSV polyprotein translational product lacked binding affinity for P150. These findings argue for differences in the mechanisms of transformation by these independently derived FeSV isolates.

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Epidermal Growth Factor; Mink; Phosphotyrosine; Protein Kinases; Rats; Retroviridae; Sarcoma Viruses, Feline; Tyrosine; Viral Proteins

We have derived a line of A431 human tumor cells infected with Rous sarcoma virus (RSV). The infected cells contain the RSV-transforming protein, pp60src, which has characteristic tyrosine specific protein kinase activity. As in other RSV-transformed cells, a 36,000-dalton protein is phosphorylated in RSV-infected A431 cells. Addition of epidermal growth factor (EGF) to the cells induces further phosphorylation of this protein. In contrast, this phosphoprotein is not detected in uninfected A431 cells, except when treated with EGF. Increased phosphorylation of the EGF receptor protein and of an 81,000-dalton cellular protein is dependent upon addition of EGF to the culture fluids, in both control and RSV-infected A431 cells. The results are discussed with reference to the similarities and differences between the tyrosine-specific protein kinases induced by RSV and activated by EGF.

Topics: Avian Sarcoma Viruses; Cell Line; Cell Transformation, Viral; Epidermal Growth Factor; ErbB Receptors; Humans; Molecular Weight; Peptide Fragments; Phosphoproteins; Phosphorylation; Phosphotyrosine; Protein Kinases; Receptors, Cell Surface; Tyrosine

Transformation by several RNA tumour viruses seems to be mediated by virally coded protein kinases which specifically phosphorylate tyrosine. A tyrosine-specific protein kinase also seems to be involved in the mitogenic action of epidermal growth factor (EGF). This EGF-stimulated kinase activity is closely associated with the EGF receptor, with which it copurifies during EGF-affinity chromatography. Because both the virus- and EGF-stimulated tyrosine kinases may be involved in stimulation of cell growth, and because the viral kinases may be antigenically related to normal cell proteins, we examined the interaction of antibodies to viral tyrosine kinases with the affinity-purified EGF receptor-kinase preparation. We report here that the receptor-kinase specifically phosphorylates antibodies directed against the transforming protein kinase pp60src of Rous sarcoma virus. However, none of these antibodies, including those which cross-react with the normal cellular homologue of pp60src (pp60sarc), precipitate the receptor-kinase. These results suggest that the EGF receptor-kinase is related to, but probably not identical with, pp60sarc.

Topics: Antibodies, Neoplasm; Avian Sarcoma Viruses; Cell Transformation, Viral; Cross Reactions; Epidermal Growth Factor; ErbB Receptors; Immunoglobulin G; Molecular Weight; Oncogene Protein pp60(v-src); Phosphoproteins; Phosphorylation; Protein Kinases; Receptors, Cell Surface; Viral Proteins

Topics: Animals; Cell Transformation, Viral; Cells, Cultured; Electrophoresis, Polyacrylamide Gel; Epidermal Growth Factor; Kidney; Kirsten murine sarcoma virus; Membrane Proteins; Mice; Molecular Weight; Peptides; Phosphates; Phosphorylation; Sarcoma Viruses, Murine; Temperature

Kirsten sarcoma virus produced a low incidence of transient morphological transformation in primary cultures of rat ovarian granulosa cells. In the presence of epidermal growth factor, the incidence of transient transformation increased severalfold and two continuous cell lines were established. Epidermal growth factor, a naturally occurring polypeptide hormone, appears to act here as a tumor promoter in the retrovirus-induced transformation of a mesodermally derived epithelium.

Topics: Animals; Cell Transformation, Viral; Cells, Cultured; Epidermal Growth Factor; Female; Granulosa Cells; Kirsten murine sarcoma virus; Peptides; Rats; Sarcoma Viruses, Murine

Cultured cell lines of human tumour origin as well as cells transformed by various RNA tumour viruses secrete low molecular weight polypeptide transforming growth factors (TGFs). In addition to competing with epidermal growth factor (EGF) for binding to its cellular receptor, TGFs can transform morphologically fibroblast and epithelial cells in culture. In view of accumulating evidence that tyrosine phosphorylation activity is associated with the transforming genes of various tumour viruses, we determined whether phosphotyrosine levels were elevated in these human tumour cells. We show here that TGFs produced by human tumour cells induce phosphorylation of specific tyrosine acceptor sites in the 160,000-molecular weight (160 K) EGF receptor.

Topics: Cell Line; Cell Transformation, Viral; Epidermal Growth Factor; ErbB Receptors; Humans; Peptides; Phosphorylation; Protein Kinases; Receptors, Cell Surface; Transforming Growth Factors; Tyrosine

Treatment of A-431 human epidermoid carcinoma cells with epidermal growth factor (EGF) was shown to enhance the phosphorylation of a Mr = 34,000 protein. Because the phosphorylation of an analogous protein is enhanced in various cell lines transformed by Rous sarcoma virus (RSV) (Erikson, E., and Erikson, R. L. (1980) Cell 21, 829-836), we characterized the phosphorylation of the A-431 Mr = 34,000 protein under these two conditions in order to determine whether there are common pathways between viral transformation and EGF stimulation. The results of tryptic phosphopeptide mapping and phosphoamino acid analysis showed that the Mr = 34,000 protein was phosphorylated in an identical manner by the EGF-stimulated protein kinase activity and by the protein kinase activity of the RSV transformation-specific protein or of its normal cell homolog. Although the specific protein kinase that phosphorylates the Mr = 34,000 protein under conditions of EGF-stimulation is not yet identified, these studies demonstrate that at least one consequence of EGF stimulation is identical with one of the consequences of viral transformation.

Topics: Avian Sarcoma Viruses; Carcinoma, Squamous Cell; Cell Line; Cell Transformation, Viral; Epidermal Growth Factor; Humans; Molecular Weight; Neoplasm Proteins; Oncogene Protein pp60(v-src); Peptide Fragments; Phosphopeptides; Phosphorylation; Trypsin; Viral Proteins

Serum-free conditioned media was collected from three sarcoma virus-transformed cell lines and an untransformed cell line. All three virally transformed lines produced and released growth factors into their serum-free media. The major activity in all cases, whether the cells were transformed by Moloney sarcoma virus (MSV) or Kirsten sarcoma virus (KiSV), or whether they were mouse or rat, was a sarcoma-growth-factor (SGF)-like activity with an apparent molecular weight of 10,000. The SGF-like pools from a Moloney sarcoma virus-transformed mouse 3T3 cell and a Kirsten sarcoma virus-transformed NRK cell were further purified by carboxymethyl cellulose chromatography. The elution profiles of these peptides were very similar. The serum-free conditioned media from the untransformed cells showed no detectable growth stimulating activity. The temperature sensitivity of an SGF-like growth factor from the supernate of a NRK cell transformed by a wild-type Kirsten sarcoma virus (KiSV) was compared with that of the SGF-like activity from the supernates of a NRK cell transformed by a ts-mutant of KiSV that is temperature sensitive with respect to transformation (ts-371 Cl 5). Neither the cells transformed by the wild-type sarcoma virus nor those transformed by the temperature sensitive virus released a SGF-like activity that was temperature sensitive under the conditions of the assays.

Topics: Animals; Cell Transformation, Viral; Cells, Cultured; Epidermal Growth Factor; Growth Substances; Mice; Neoplasm Proteins; Sarcoma Viruses, Murine; Temperature

Many conditions affect the growth of animal cells in culture. Some, such as essential nutrients, are necessary for growth as precursors of macromolecules. Other substances appear to have regulatory functions. Their presence or absence determines whether the cell will continue to grow or move into a resting, quiescent state. In particular certain factors (usually provided by serum) are thought to have regulatory roles. To decide which factors are regulatory is a difficult problem. We propose here that no single criterion is sufficient to permit a decision and that the best course at present is to apply several criteria. Three such criteria are proposed. To the degree that the tests are satisfied we can tentatively decide whether or not a given factor is regulatory.

Topics: Animals; Caffeine; Cell Cycle; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cells, Cultured; Cycloheximide; Epidermal Growth Factor; Humans; Insulin; Kinetics; Lectins; Mutation; Tetradecanoylphorbol Acetate; Thrombin

Topics: Animals; Cell Line; Cell Transformation, Viral; Epidermal Growth Factor; Glycoproteins; Kirsten murine sarcoma virus; Mice; Phorbols; Protein Biosynthesis; RNA, Messenger; Sarcoma Viruses, Murine; Tetradecanoylphorbol Acetate; Tretinoin

Topics: Animals; Carcinoma; Cell Line; Cell Transformation, Viral; Epidermal Growth Factor; Growth Substances; Humans; Kinetics; Mice; Peptides; Receptors, Cell Surface; Sarcoma Viruses, Murine; Submandibular Gland; Transforming Growth Factors

Glucocorticosteroids, when added two hours after cell plating to SV40-transformed, 3T3 mouse fibroblasts in low serum (0.3% v/v), biotin-supplemented medium, suppress cellular proliferation by 24 hours. While some cell death probably occurs, the growth inhibition is not primarily due to cytotoxicity and cytolysis. This conclusion is supported by the following: 1) both dead and viable cell numbers are suppressed, 2) little cell debris is evident in the medium, and 3) very high concentrations of glucocorticoids do not cause an increase in the dead cell count. Furthermore, this growth suppression, which is specific for glucocorticoids since several non-glucocorticoid steroids have no inhibitory effect, is not permanent nor irreversible. Removal of the glucocorticoid and replacement with 10% serum restore rapid proliferation. Although higher concentrations (1% and 10%) of serum afford some protection against glucocorticoid inhibition, this protection is not simply a consequence of faster growth rates. SV3T3 cells can be grown in serum-free medium supplemented with biotin, transferrin, insulin, and epidermal growth factor (EGF). Under these conditions growth rates are comparable to high serum media, yet glucocorticoids are still powerful inhibitors. However, the omission of insulin from serum-free, glucocorticoid cultures does result in observable cell death and lysis. Flow microfluorometry and autoradiographic studies have determined that glucocorticoid-inhibited cells are partially blocked in G1. The proportions of S phase and G2 + M cells are greatly reduced with an accompanying accumulation of G1 cells. These results suggest that glucocorticoids regulate a biochemical step(s) in G1 which is critical for DNA initiation.

Topics: Animals; Blood; Cell Division; Cell Line; Cell Transformation, Viral; Dexamethasone; Epidermal Growth Factor; Interphase; Mice; Prednisolone; Simian virus 40; Steroids; Structure-Activity Relationship; Triamcinolone

Cells transformed by murine sarcoma viruses (MSV) produce and release into their tissue culture media several polypeptide growth stimulating factors. One of these has been partially purified using Bio-Gel P-60 column chromatography followed by DEAE-cellulose chromatography. This growth factor was assigned the name sarcoma growth factor (SGF), and is here shown to require the epidermal growth factor (EGF) receptor in order to function as a growth factor. DEAE-cellulose chromatography yielded a product that was several-fold purer than the material present in the Bio-Gel P-60 column pool II. The biologically active material from the DEAE-cellulose column, when labeled with 125I, showed specific binding to EGF membrane receptors. The specific binding could be prevented with the addition of either unlabeled EGF or SGF. Both radiolabeled SGF and EGF will bind to live or fixed cells. We were able to bind 125I-SGF as well as 125I-EGF to fixed cells and elute the bound material from fixed receptors. The eluted SGF showed a greater than 25-fold increase in specific binding. The biological activities of EGF and SGF could be bound to and eluted from fixed receptors. The eluted SGF showed a greater than 25-fold increase in specific binding. The biological activities of EGF and SGF could be bound to and eluted from fixed cells. A 3T3 clone lacking EGF receptors was unable to respond to either EGF or SGF, whereas it responded well to serum and several other purified growth factors. The SGF isolated using DEAE-cellulose chromatography was unable to compete in a radioimmune assay using 125I-EGF and antibody to purified mouse submaxillary gland EGF; it also was not precipitated by anti-EGF antibody. From these studies it appears that the SGF produced and released by these MSV-transformed cells combines with and requires the EGF receptor in order to exert its biological effects. The peptide, however, is antigenically distinct from mouse submaxillary gland EGF.

Topics: Animals; Antigens; Cell Division; Cell Line; Cell Transformation, Viral; DNA; Epidermal Growth Factor; Growth Substances; Mice; Peptides; Receptors, Drug; Sarcoma Viruses, Murine

Topics: Adenoviruses, Human; Animals; Carcinogens; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cells, Cultured; Epidermal Growth Factor; Mice; Peptides; Phorbols; Rats; Receptors, Cell Surface; Tetradecanoylphorbol Acetate

Malignant transformation by mammalian RNA sarcoma viruses has previously been shown to involve a reduction in receptor sites for a well characterized 6,000-molecular weight (MW) growth-promoting substance, designated epidermal growth factor (EGF). Although Abelson murine leukaemia virus (AbLV) resembles sarcoma viruses in its ability to transform embryo fibroblasts in cell culture, AbLV induces a rapid B-cell lymphoid leukaemia rather than fibrosarcomas in vivo. The major translational product of AbLV is a highly phosphorylated polyprotein of MW 120,000 which exhibits an associated tyrosine-specific protein kinase activity and probable transforming function. We show here that AbLV transformation resembles transformation by RNA sarcoma viruses with respect to the abolition of EGF-binding sites. EGF binding is restored to control levels following loss of polyprotein expression in morphological revertants of AbLV-transformed clones and remains uninfluenced in cell lines infected with transformation-defective (td) AbLV mutants encoding polyproteins deficient in protein kinase activity. These findings indicate that AbLV transformation involves a polyprotein-associated, tyrosine-specific protein kinase activity which mediates its effect through a mechanism resulting directly or indirectly in the abolition of EGF-binding sites.

Topics: Abelson murine leukemia virus; Animals; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Epidermal Growth Factor; Leukemia Virus, Murine; Mink; Peptides; Phosphoproteins; Protein Kinases; Rats; Receptors, Cell Surface; Tyrosine

Topics: Affinity Labels; Animals; Cell Line; Cell Transformation, Viral; Embryo, Mammalian; Epidermal Growth Factor; Gastrointestinal Hormones; Kinetics; Mice; Molecular Weight; Peptides; Receptors, Cell Surface; Simian virus 40; Thymidine

We have examined culture fluids from a variety of Kirsten murine sarcoma virus (KiMSV) transformed rat and mouse cells for the presence of factors which induce normal Rat-1 cells to assume the transformed phenotype. All KiMSV transformants produced transforming factor (TF). Revertants of KiMSV transformed rat or mouse failed to release TF as did normal rat or mouse cells. Cells transformed by a temperature sensitive mutant of KiMSV produced TF at the permissive temperature but not at the nonpermissive temperature. Further, cells from a spontaneous transformant of Rat-1 cells also produced TF. TF is a small polypeptide which competes for the epidermal growth factor receptor. Its effect upon normal cells is reversible and requires de novo RNA and protein synthesis. Cells treated with TF lose the actin fibers observed in normal fibroblasts, assume a transformed cell morphology, become anchorage independent for growth, grow in low concentrations of serum, grow to a high cell density, and have an increased rate of hexose uptake.

Topics: Actins; Animals; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Deoxyglucose; Epidermal Growth Factor; ErbB Receptors; Kirsten murine sarcoma virus; Peptide Biosynthesis; Rats; Receptors, Cell Surface; Transforming Growth Factors

Griseofulvin, 12-O-tetradecanoyl phorbol-13-acetate, melittin, epidermal growth factor, vinblastine, cytochalasin B, podophyllotoxin, colcemid, and colchicine were unable to transform cells but could increase from 8- to 40-fold the frequency of cell transformation by polyoma virus. The 3T3-like cells were resting at confluence and were exposed to the drug only during the 1st week after viral infection. Griseofulvin, a tumor promoter, reduced or increased the frequency of transformation depending on the dose with which the infected cells were treated. The antitumor activity of tumor promoters is discussed.

Topics: Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Colchicine; Cytochalasin B; Epidermal Growth Factor; Griseofulvin; Melitten; Mutagens; Podophyllotoxin; Polyomavirus; Tetradecanoylphorbol Acetate; Vinblastine

Topics: Animals; Cell Transformation, Viral; Cells, Cultured; DNA Replication; Epidermal Growth Factor; Extracellular Space; Glycoproteins; Growth Substances; Kinetics; Methionine; Mice; Mitogens; Molecular Weight; Protein Biosynthesis

Monolayer cultures of human prostatic epithelial cells were exposed to SV40 virus at 35th population doubling. Clones were isolated from infected plates after growth had ceased on the control plates. The nuclei of these clones were virtually all positive for viral T-antigen by immunofluorescence. When the properties of three of these lines were compared to those of normal cells, they were found to have altered morphology, ultrastructure, chromosomes, and growth behavior. All transformed lines had reduced serum dependence and were capable of growing in soft agar. However, their reduced serum dependence was not due to reduced growth factor requirements because each subline's response to growth factors was different.

Topics: Agar; Antigens, Polyomavirus Transforming; Blood Physiological Phenomena; Calcium; Cell Division; Cell Size; Cell Transformation, Viral; Clone Cells; Culture Media; Epidermal Growth Factor; Epithelial Cells; Fibroblast Growth Factors; Fibroblasts; Growth Substances; Humans; Karyotyping; Magnesium; Male; Microscopy, Electron; Ploidies; Prostate; Simian virus 40

Serum provides growth factors that regulate and limit the growth of normal cells in tissue culture. Animal cells that are malignantly transformed usually exhibit diminished serum requirements for growth in culture. We have used a defined, serum-free medium to determine which of these growth factors becomes dispensable for the growth of transformed Syrian and Chinese hamster fibroblast cells. The medium's four growth factors-epidermal growth factor (EGF), insulin, fibroblast growth factor, and transferrin-were added or omitted as desired. A decreased requirement for EGF was most closely related to tumorigenicity of chemically (ethyl methanesulfonate) transformed cells in nude mice. All lines examined retained their requirement for transferrin, which is needed throughout the growth cycle, in contrast to the other factors, which are needed primarily in G(1) phase. Lines that had lost their EGF requirement but had retained their insulin requirement were arrested in G(1) by insulin deficiency, indicating that their growth control system remained. Mutagenesis with ethyl methanesulfonate can also create requirements of the transformed cells for unknown factors in serum. We conclude that an initial step that reduces the serum requirement in culture, and in tumorigenesis, is relaxation of the growth-regulatory function of EGF.

Topics: Blood; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Culture Media; Epidermal Growth Factor; Fibroblasts; Growth Substances; Insulin; Peptides; Polyomavirus; Transferrin

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Epidermal Growth Factor; Humans; Neoplasms; Peptides; Protein Biosynthesis; Receptors, Cell Surface

Murine sarcoma virus-transformed mouse fibroblasts produce polypeptide growth factors and release them into serum-free medium. These factors stimulate cells to divide in monolayer cultures and also to form colonies that grow progressively soft agar. Three major peaks of activity are seen, with apparent molecular weights of 25,000, 12,000, and 7000. The sarcoma growth factors are heat-stable, trypsin-sensitive, and active in nanogram quantities when tested for growth stimulation of untransformed rat and mouse fibroblasts. All three molecular species are also capable of competing for membrane epidermal growth factor (EGF) receptors when tested with 125I-labeled EGF. They differ from mouse EGF, however, in their molecular weights, in their inability to react with anti-EGF antibodies, and in their ability to convert cells to anchorage independent (agar) growth. For the above reasons, we conclude that the sarcoma growth factors are a new class of polypeptide tropic factors that confer on fibroblasts in vitro properties associated with the transformed phenotype.

Topics: Binding Sites; Binding, Competitive; Cell Division; Cell Transformation, Viral; Cells, Cultured; Epidermal Growth Factor; Gammaretrovirus; Growth Substances; Hot Temperature; Molecular Weight; Moloney murine leukemia virus; Sarcoma Viruses, Murine; Trypsin

Topics: Animals; Benzopyrenes; Blood Proteins; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cycloheximide; Dactinomycin; Epidermal Growth Factor; Mice; Plasminogen Activators; Simian virus 40

Fibroblastic and epithelioid clones have been isolated from the normal rat kidney line, NRK. These clones were studied for their ability to bind epidermal growth factor (EGF), susceptibility to transformation by mouse sarcoma virus (MSV), and alteration in EGF binding upon sarcoma virus transformation. The epithelioid clones bound much more EGF than the fibroblastic clones; Scatchard plots on two of these clones, one epithelioid and one fibroblastic, showed that the higher EGF binding (1.3 x 10(5) molecules per cell for the epithelioid clone and 1.3 x 10(4) molecules per cell for the fibroblastic clone) was due to a greater number or receptors on the epithelioid cells rather than to a difference in the apparent affinity constant. When the clones were transformed by Moloney murine sarcoma virus the EGF binding decreased, the effect being greater with the fibroblastic clones. In 20 out of 20 independently isolated sarcoma virus transformed fibroblastic clones, the level of EGF binding was either greatly reduced or completely eliminated. In contrast to EGF, another growth factor, multiplication stimulating activity (MSA), bound to a greater extent to the fibroblastic clones than the epithelioid clones, and its binding was not decreased by sarcoma virus transformation. The results show that loss of EGF binding ability correlates with expression of the murine sarcoma virus transformation.

Topics: Animals; Cell Line; Cell Transformation, Viral; Clone Cells; Epidermal Growth Factor; Epithelial Cells; Fibroblasts; In Vitro Techniques; Mice; Moloney murine leukemia virus; Peptides; Submandibular Gland

Topics: Blood; Calcium; Cell Cycle; Cell Line; Cell Transformation, Viral; DNA; Epidermal Growth Factor; Ornithine Decarboxylase; Peptides; Polyamines; Receptors, Drug; RNA, Ribosomal

Topics: Cell Division; Cell Transformation, Viral; Cells, Cultured; Cycloheximide; Epidermal Growth Factor; Fluorides; Mitogens; Peptides; Receptors, Drug; Temperature; Thymidine