methylnitronitrosoguanidine and Cell-Transformation--Neoplastic

Topics: 9,10-Dimethyl-1,2-benzanthracene; Actins; Animals; Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; Cocarcinogenesis; DNA; Gene Expression Regulation, Neoplastic; Matrix Metalloproteinase 3; Metalloendopeptidases; Methylnitronitrosoguanidine; Mice; Neoplasm Metastasis; Neoplasm Proteins; Neoplasms, Multiple Primary; Papilloma; Skin Neoplasms; Tetradecanoylphorbol Acetate; Ubiquitins

Expression of TGF-alpha mRNA, which correlates well with the ability of cells to condition medium with an EGF-like activity, clonally segregates best with tumorigenicity among the several single phenotypes considered in this study. The results of unreported studies in which we have analyzed the quantitative relationships between the expression of selected phenotypes and tumorigenicity, suggest that the elevated expression of myc and TGF-alpha mRNAs interact in their associations with tumor yield. These results suggest that elevated myc expression sensitizes hepatic epithelial cells to the possible tumorigenic action of TGF-alpha. This observation may explain why the correlation between the qualitative expression of TGF-alpha and tumorigenicity, described here, is not perfect. Conventionally applied markers of transformation in hepatocytes in vivo and in cultured liver epithelial cells in vitro that we studied -histochemical expression of GGT, ability to grow in medium containing low levels of calcium, and ability to grow in soft agar- clonally segregated with tumorigenicity poorly in liver epithelial cells transformed in vitro. We conclude that these phenotypes are not adequate markers for determining the lineage of hepatic epithelial neoplasms (including, probably hepatocellular cancers arising in vivo). This study appears to be the first to attempt to analyze clonally the association of these markers with tumorigenicity, and to quantify the sensitivity, specificity, and predictive value of the associations. Our study suggests that the relatively weak associations of these phenotypes with tumorigenicity may be related only to their stronger associations with expression of TGF-alpha, or to some other property that is strongly associated with tumorigenicity. Expression of TGF-alpha is more strongly associated with expression of GLC, for example, than is the GLC phenotype with tumorigenicity. At least for GLC, autocrine stimulation by TGF-alpha is likely, since EGF increases growth of WB cells in low calcium medium. This observation may explain the perfect correlation between expression of TGF-alpha and GLC. EGF also stimulates lactate dehydrogenase, pyruvate kinase, and glucose 6-phosphate dehydrogenase in WB cells. However, quantitative correlation between GGT activity and TGF-alpha is less strong. Thus, our data from these studies suggest that the tumorigenic phenotype of cultured hepatic epithelial cells is intimately dependent on the expression of the

Topics: Animals; Biomarkers, Tumor; Cell Line, Transformed; Cell Transformation, Neoplastic; Clone Cells; DNA, Neoplasm; Epithelium; Gene Expression Regulation, Neoplastic; Liver; Methylnitronitrosoguanidine; Mice; Mice, Nude; Neoplasm Proteins; Neoplasm Transplantation; Phenotype; Rats; Rats, Inbred F344

Diploid WB rat liver epithelial cells contain abundant, rapidly internalized epidermal growth factor receptors, and respond pleiotropically to ligand binding. Signal transduction pathways downstream from the EGF receptor involve activation of elements that are both dependent on and independent of protein kinase C activation. Neoplastic transformation of wild-type WB rat liver epithelial cells by exposure to N-methyl-N'-nitro-N-nitrosoguanidine is associated with progressive alterations in the responses of affected cells to binding of EGF to EGF receptors, including heightened cell proliferation and the expression of several other phenotypic properties. Tumorigenic rat liver epithelial cells acquire the ability to express transforming growth factor-alpha (TGF-alpha), and to secrete this growth factor in a regulated and then unregulated manner. TGF-alpha expression, together with the presence of abundant EGF receptors, provides affected cells with an autocrine growth cycle. The ability of transformed WB rat liver epithelial cells to produce tumors cosegregates clonally with TGF-alpha expression and with heightened expression of c-myc, c-Ha-ras and c-Ki-ras proto-oncogenes.

Topics: Animals; Cell Line; Cell Line, Transformed; Cell Transformation, Neoplastic; Epidermal Growth Factor; Epithelium; ErbB Receptors; Gene Expression Regulation, Neoplastic; Liver; Methylnitronitrosoguanidine; Rats; Transforming Growth Factors

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Cell Differentiation; Cell Line, Transformed; Cell Transformation, Neoplastic; Cells, Cultured; Methylnitronitrosoguanidine; Mice; Skin Neoplasms; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Topics: Animals; Carcinogens; Cell Transformation, Neoplastic; Disease Models, Animal; Epithelium; Gastric Mucosa; Humans; Methylnitronitrosoguanidine; Mice; Mucus; Precancerous Conditions; Rats; Stomach; Stomach Neoplasms

Topics: Animals; Biotransformation; Carcinogens; Cell Line; Cell Transformation, Neoplastic; Clone Cells; Methyl Methanesulfonate; Methylnitronitrosoguanidine; Mice; Mice, Inbred BALB C; Research Design

Topics: Animals; Carcinogens; Cell Line; Cell Transformation, Neoplastic; Humans; Methylnitronitrosoguanidine; Mice; Neoplasms, Experimental

Since the discovery of the mutagenic activity of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in 1960, this compound has become one of the most widely used chemical mutagens. The present paper gives a survey on the chemistry, metabolism, and mode of interaction of MNNG with DNA and proteins, and of the genotoxic effects of this agent on microorganisms, plants, and animals, including human cells cultured in vitro. Data on the carcinogenicity and teratogenicity of MNNG as well as on the genotoxic effects of homologs of MNNG are also presented.

Topics: Animals; Bacteria; Bacteriophages; Carcinogens; Cell Transformation, Neoplastic; DNA; DNA Repair; DNA Replication; Fungi; Methylnitronitrosoguanidine; Mutagens; Mutation; Plasmids; Proteins; Teratogens; Viruses

Topics: 4-Nitroquinoline-1-oxide; Animals; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cells, Cultured; Cricetinae; DNA Repair; Humans; Methylnitronitrosoguanidine; Mice; Mitomycins; Mutation; Ultraviolet Rays

We propose that SF derived from normal-appearing biopsies of ACR gene carriers exist in an initiated state as the result of a dominant mutation. Based on our studies with the ACR cell system, we further suggest that, although an initiated state is essential to cancer development, not all initiated cells necessarily develop into cancerous cells. The genetic makeup of an initiated cell has been established through linkage between abnormal phenotypic markers and pedigree profiles and through cell hybridization, including initial analysis of gene products. We believe that it is consistent with an autosomal dominant trait. In contrast, cells from patients who are homozygous for chromosomal breakage syndromes, including those with xeroderma pigmentosum, represent an experiment of nature which presumably underlies factors associated with cancer promotion in humans. We have demonstrated that ACR cells can be differentially transformed by oncogenic viruses, a carcinogen (MNNG), and gamma-ray irradiation, and that they can proliferate in vitro after exposure to a tumor promoter (TPA. This simple experimental model provides a novel system for the study of tumor promotion in vitro. We further suggest that, through the use of TPA, various stages associated with cancer development in humans, i.e., initiation through promotion and progression, can be identified in vitro. Attempts to apply these results in vivo are currently in progress. The apparent susceptibility of ACR cells to further transformation by oncogenic viruses and chemical and physical agents indicates that genetic information residing within these cells, probably in the form of a relatively limited and specific number of DNA sequences associated with the ACR mutation, renders them more sensitive to these three distinct classes of carcinogens. We submit that, through our tests on SF, and ACR gene carriers within recognized ACR clusters can be diagnosed at present with sufficient certainty to warrant immediate action. In addition, it seems that the time has arrived for a major undertaking to screen for persons who are likely to be at increased risk of cancer, perhaps through walk-in clinics. An underlying assumption in these studies is that predisposition to cancer, in general, is associated with an autosomal dominant trait in obligatory heterozygote gene carriers.

Topics: Actins; Adenoma; Antigens, Neoplasm; Carcinogens; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cells, Cultured; Cholesterol; Cocarcinogenesis; Colonic Neoplasms; Cytoskeleton; Disease Susceptibility; Genes, Dominant; Humans; Kirsten murine sarcoma virus; Methylnitronitrosoguanidine; Mitochondria; Models, Biological; Mutation; Plasminogen Activators; Prognosis; Rectal Neoplasms

Topics: Adenoviruses, Simian; Animals; Carcinogens; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cells, Cultured; Chromosome Aberrations; Chromosome Banding; Chromosomes; Cricetinae; DNA; Heterochromatin; Karyotyping; Mesocricetus; Methylnitronitrosoguanidine; Sex Chromosomes

Chromosomal abnormalities are a frequent concomitant of neoplasia, and although it is tempting to relate these mutations and alterations in chromatin (DNA) function to cancer, their relationship to the initiation or progression of carcinogenesis is unknown. Mammalian cells in culture, after interacting with chemical carcinogens, often exhibit chromosome damage consisting of breaks and exchanges of chromatid material. The pattern of damage of banded metaphases indicates that negative bands are especially vulnerable to the action of chemical carcinogens, probably because of differential chromatin condensation. Damage to individual chromosomes may be random or nonrandom, depending on the species. Cell death can be correlated with chromatid alterations that occur shortly after treatment with chemical carcinogens. There is also a correlation between mutagenic and carcinogenic activity of some chemical carcinogens and the frequency of sister chromatid exchanges. The question of whether specific chromosome changes are absolutely required for neoplastic transformation cannot be answered because of conflicting data and diverse results from studies even with known carcinogens. Cell transformation may occur without any visible chromosome changes. A universal specific numerical or visible structural chromosomal alteration is not necessarily associated with chemical or viral transformation. Chromosome changes are independent of the etiologic agents: different carcinogens may produce transformation associated with the same abnormal chromosomes, but not all transformed lines invariably exhibit the same abnormality, even with the same chemical. In some species, chromosome having nucleolar organizer regions may be more frequently involved in numerical or structural deviations. Progressively growing tumors also may occur as a result of the proliferation of transformed cells without detectable chromosome changes, indicating that tumorigenicity need not be related to an imbalance of chromosome number or structure. Our studies indicate that chromosome changes are not essential for establishment of neoplasms but that karyotypic instability may result in response to selective growth pressures.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Azure Stains; Carcinogens; Cell Line; Cell Nucleolus; Cell Transformation, Neoplastic; Chromatids; Chromosomes; Humans; Karyotyping; Methylnitronitrosoguanidine; Neoplasms; Transformation, Genetic; Trisomy

Hepatocellular carcinoma is a malignancy with poor clinical prognosis. Hepatic oval cells (HOCs) tend to differentiate into cancerous hepatocellular carcinoma cells (HCCs) in the tumor microenvironment. The purpose of the present study was to explore the role of kangxianruangan granule (KXRG)‑containing serum in inhibiting the differentiation of HOCs into HCCs via the Wnt‑1/β‑catenin signaling pathway. N‑methyl‑N'‑nitro‑N‑nitrosoguanidine (MNNG) was applied to induce the transformation of the rat HOC cell line WB‑F344 into HCCs. The overexpression plasmid, Wnt‑1‑up, was utilized to increase Wnt‑1 expression. Subsequently, high, medium and low concentrations of KXRG were applied to MNNG‑treated WB‑F344 cells to assess the inhibitory effect of KXRG on cell differentiation. Flow cytometry was conducted to detect the cell cycle distribution, apoptotic rate and expression of cytokeratin‑19 (CK‑19) protein in cells. An immunofluorescence double staining protocol was used to detect the expression of Wnt‑1 and β‑catenin. ELISAs were performed to detect α fetoprotein in the cell supernatants. Reverse transcription‑quantitative PCR and western blotting were conducted to detect the mRNA and protein expression levels of Wnt‑1, β‑catenin, Cyclin D1, C‑myc, matrix metalloproteinase‑7 (MMP‑7), Axin2 and epithelial cell adhesion molecule (EpCAM) in cells. Compared with the normal group, the apoptotic rate, proportion of S phase cells, concentration of AFP in the cell supernatant, level of CK‑19 protein, and mRNA and protein expression levels of Wnt‑1, β‑catenin, Cyclin D1, C‑myc, MMP‑7, Axin2 and EpCAM were all significantly increased in the model group. Addition of KXRG significantly reduced the aforementioned indicators compared with the model group. Moreover, Wnt‑1 overexpression further increased the aforementioned indicators compared with the model group, whereas KXRG significantly inhibited these effects. The results indicated that KXRG inhibited the differentiation of HOCs into HCCs via the Wnt‑1/β‑catenin signaling pathway, which suggested the potential clinical application of KXRG for the prevention of hepatocellular carcinoma.

Topics: Animals; Carcinoma, Hepatocellular; Cell Differentiation; Cell Line, Tumor; Cell Transformation, Neoplastic; Disease Models, Animal; Drugs, Chinese Herbal; Humans; Liver; Liver Neoplasms, Experimental; Male; Methylnitronitrosoguanidine; Rats; Tumor Microenvironment; Wnt Signaling Pathway

Studies have shown that environmental carcinogens exerted an important function in the high incidence of esophageal cancer (EC). Nitrosamines have been identified as important environmental carcinogens for EC. This study aimed to investigate the metabolic disturbances and new key toxicological markers in the malignant transformation process of normal esophageal epithelial cells (Het-1A) induced by MNNG (N-methyl-N'-nitro-N-nitrosoguanidine). Untargeted metabolomic and lipidomic profiling analysis by using ultra-high-performance liquid chromatography coupled with mass spectrometry (UHPLC-MS) were applied to explore the metabolic network alterations of Het-1A cells. The metabolomic results showed that significant alterations were observed in metabolic signatures between different generations (P5, P15, P25, P35) and the control cell group (P0). A total of 48 differential endogenous metabolites were screened and identified, mainly containing fatty acids, amino acids, and nucleotides. The differential metabolites were predominantly linked to the pathway of biosynthesis of unsaturated fatty acids metabolism. The cell lipidomic profiling revealed that the most differential lipids contained fatty acids (FAs), phosphatidylcholines (PC), phosphatidylethanolamines (PE), and phosphatidylserines (PS). The enrichment of the lipidomic pathway also confirmed that the lipid metabolism of biosynthesis of unsaturated fatty acids was the significant variation during the cell malignant transformation. Furthermore, we detected the expression of the upstream regulatory enzymes related to the unsaturated fatty acids to explore the regulation mechanism. The expression of stearoyl-CoA desaturase (SCD), ELOVL fatty acid elongase 1 (ELOVL1) promoted, and fatty acid desaturase 1 (FADS1) inhibited the key fatty acids of unsaturated fatty acids metabolism compared to the control cell group. Overall, our results revealed that lipid fatty acid metabolism was involved in the malignant transformation of Het-1A cells induced by MNNG and deepened the awareness of the carcinogenic mechanism of environmental exposure pollutants.

Topics: Carcinogens, Environmental; Cell Transformation, Neoplastic; Esophageal Neoplasms; Fatty Acids; Fatty Acids, Unsaturated; Humans; Metabolomics; Methylnitronitrosoguanidine

N-methyl-N´-nitro-N-nitrosoguanidine is a clear carcinogen, increasing evidence that indicates an etiological role of human papillomavirus in esophageal carcinoma. Studies have reported the synergistic effect on environmental carcinogens and viruses in recent years. On the basis of establishing the malignant transformation model of Het-1A cells induced by synergistic of HPV18 and MNNG, this study was to explore the synergistic carcinogenesis of MNNG and HPV. Our research indicated that HPV&MNNG led to a significant increase in the protein-expression levels of c-Myc, cyclinD1, BCL-2, BAX, E-cadherin, N-cadherin, mTOR, LC3II, and p62, with concomitant decreases in p21 and LC3I. HPV18 and MNNG induced accumulation of p62 and its interaction with KEAP1, which promoted NRF2 nuclear translocation. p62 loss prevents growth and increases autophagy of malignant cells by activating KEAP1/NRF2-dependent antioxidative response. In addition, PI3K and p-AKT were stimulated by HPV&MNNG, and PI3K/AKT/mTOR is positively associated with cell proliferation, migration, invasion, and autophagy during malignant transformation. Taken together, MNNG&HPV regulates autophagy and further accelerates cell appreciation by activating the p62/KEAP1/NRF2 and PI3K/AKT/mTOR pathway. MNNG&HPV may improve Het-1A cell autophagy to contribute to excessive cell proliferation, reduced apoptosis, and protection from oxidative damage, thus accelerating the process of cell malignant transformation and leading to cancerous cells.

Topics: Autophagy; Cell Proliferation; Cell Transformation, Neoplastic; DNA-Binding Proteins; Epithelial Cells; Gene Expression Regulation; Humans; Kelch-Like ECH-Associated Protein 1; Methylnitronitrosoguanidine; NF-E2-Related Factor 2; Oncogene Proteins, Viral; Oxidative Stress; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; RNA Interference; RNA, Small Interfering; Sequestosome-1 Protein; Signal Transduction; TOR Serine-Threonine Kinases

Study shows that signal transducer and activator of transcription 3 (STAT3) can increase the Warburg effect by stimulating hexokinase 2 in breast cancer and upregulate lactate dehydrogenase A and pyruvate dehydrogenase kinase 1 in myeloma. STAT3 and pyruvate kinase M2 (PKM2) can also be activated and enhance the Warburg effect in hepatocellular carcinoma. Precancerous lesions are critical to human and rodent hepatocarcinogenesis. However, the underlying molecular mechanism for the development of liver precancerous lesions remains unknown. We hypothesized that STAT3 promotes the Warburg effect possibly by upregulating p-PKM2 in liver precancerous lesions in rats.. To investigate the mechanism of the Warburg effect in liver precancerous lesions in rats.. A model of liver precancerous lesions was established by a modified Solt-Farber method. The liver pathological changes were observed by HE staining and immunohistochemistry. The transformation of WB-F344 cells induced with N-methyl-N'-nitro-N-nitrosoguanidine and hydrogen peroxide was evaluated by the soft agar assay and aneuploidy. The levels of glucose and lactate in the tissue and culture medium were detected with a spectrophotometer. The protein levels of glutathione S-transferase-π, proliferating cell nuclear antigen (PCNA), STAT3, and PKM2 were examined by Western blot and immunofluorescence.. We found that the Warburg effect was increased in liver precancerous lesions in rats. PKM2 and p-STAT3 were upregulated in activated oval cells in liver precancerous lesions in rats. The Warburg effect, p-PKM2, and p-STAT3 expression were also increased in transformed WB-F344 cells. STAT3 activation promoted the clonal formation rate, aneuploidy, alpha-fetoprotein expression, PCNA expression, G1/S phase transition, the Warburg effect, PKM2 phosphorylation, and nuclear translocation in transformed WB-F344 cells. Moreover, the Warburg effect was inhibited by stattic, a specific inhibitor of STAT3, and further reduced in transformed WB-F344 cells after the intervention for PKM2.. The Warburg effect is initiated in liver precancerous lesions in rats. STAT3 activation promotes the Warburg effect by enhancing the phosphorylation of PKM2 in transformed WB-F344 cells.

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Cyclic S-Oxides; Disease Models, Animal; Glycolysis; Hepatocytes; Humans; Hydrogen Peroxide; Liver; Liver Neoplasms; Male; Methylnitronitrosoguanidine; Phosphorylation; Precancerous Conditions; Pyruvate Kinase; Rats; Rats, Wistar; STAT3 Transcription Factor; Stem Cells; Up-Regulation

We examined the role of DNA methyltransferase 1 (DNMT1) in

Topics: Animals; Carcinogens; Cell Line; Cell Transformation, Neoplastic; DNA (Cytosine-5-)-Methyltransferase 1; Epithelial Cells; Esophageal Neoplasms; Esophagus; Female; Humans; Methylnitronitrosoguanidine; Mice, Inbred BALB C; Mice, Nude

Evidence shows that aberrant expression of long non-coding RNA (lncRNA) is closely associated with tumor development and progression. However, the role of lncRNA in environmental carcinogen induced gastric tumorigenesis remains largely unknown. This study aimed at investigating the function role of lncRNA in N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) induce malignantly transformed human gastric epithelial cells.. In this study, high-throughput sequencing and qRT-PCR assay revealed marked downregulation of lncRNA LOC101927497 in the malignant transformed gastric epithelial cells induced by MNNG (GES-1-T cells), gain-of-function and loss-of-function assays showed that LOC101927497 can suppress the proliferation and migration of GES-1-T cells in vitro. RNA antisense purification experiment showed that LOC101927497 interacted with miR-574-5p in GES-1-T cells the most obvious. Further studies suggested that LOC101927497 may function as a tumor suppressor by interacting with miR-574-5p.. LncRNA LOC101927497 functions as a suppressor by interacting with miR-574-5p, thus inhibiting the malignant phenotype of GES-1-T cells.. To the best of our knowledge, this is the first study to demonstrate the role of lncRNA in MNNG-induced gastric tumorigenesis, and it will provide new insights into the role of lncRNA in environmental carcinogen-induced gastric cancer.

Topics: Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Epithelial Cells; Gastric Mucosa; Humans; Methylnitronitrosoguanidine; RNA, Long Noncoding; Stomach Neoplasms

Several possible mechanisms have been examined to gain an understanding on the carcinogenic properties of lead, which include among others, mitogenesis, alteration of gene expression, oxidative damage, and inhibition of DNA repair. The aim of the present study was to explore if low concentrations of lead, relevant for human exposure, interfere with Ape1 function, a base excision repair enzyme, and its role in cell transformation in Balb/c-3T3. Lead acetate 5 and 30 μM induced APE1 mRNA and upregulation of protein expression. This increase in mRNA expression is consistent throughout the chronic exposure. Additionally, we also found an impaired function of Ape1 through molecular beacon-based assay. To evaluate the impact of lead on foci formation, a Balb/c-3T3 two-step transformation model was used. Balb/c-3T3 cells were pretreated 1 week with low concentrations of lead before induction of transformation with n-methyl-n-nitrosoguanidine (MNNG) (0.5 μg/mL) and 12-O-tetradecanoylphorbol-13-acetate (TPA) (0.1 μg/mL) (a classical two-step protocol). Morphological cell transformation increased in response to lead pretreatment that was paralleled with an increase in Ape1 mRNA and protein overexpression and an impairment of Ape1 activity and correlating with foci number. In addition, we found that lead pretreatment and MNNG (transformation initiator) increased DNA damage, determined by comet assay. Our data suggest that low lead concentrations (5, 30 μM) could play a facilitating role in cellular transformation, probably through the impaired function of housekeeping genes such as Ape1, leading to DNA damage accumulation and chromosomal instability, one of the most important hallmarks of cancer induced by chronic exposures.

Topics: Animals; BALB 3T3 Cells; Carcinogens, Environmental; Cell Proliferation; Cell Survival; Cell Transformation, Neoplastic; Comet Assay; DNA Damage; DNA-(Apurinic or Apyrimidinic Site) Lyase; Gene Expression; Humans; Lead; Methylnitronitrosoguanidine; Mice; Models, Biological; Tetradecanoylphorbol Acetate

In recent years, Chinese medicine has played an important role in the prognosis of gastric cancer. Precancerous lesions of gastric carcinoma (PLGC) is a class of gastric cancer which is closely related to the gastric mucosal pathology changes in the role of carcinogenic incentives, and plays key role in the progression of normal gastric mucosal cells into gastric cancerous cells. In current experiment, we explore the relationship between Chinese traditional medicine (Xiao Tan He Wei Decoction) and gastric cancer in the PLGC rat animal models and epithelial-mesenchymal transitioned GES-1 cells which were induced useing 1- Methyl-3-nitro-1-nitrosoguanidine (MNNG). PLGC rat model showed significant deterioration in the gastric mucosa with terrible growth rate in body weight and more atypical hyperplasia in gastric mucosa. MC cells, MNNG induced GES-1 cells which epithelial- mesenchymal-transition (EMT)-related proteins have a great change compare with normal GES-1 cells. The cells had characteristics of malignant cells including proliferation, invasion and metastasis ability. Our research founds that Xiao Tan He Wei Decoction could inhibit cell proliferation and increased apoptosis by increase the level of pro-apoptotic proteins like Bax and caspase-3 and decreased the level of anti-apoptotic protein Bcl-2, block the cells in G0/G1 phase simultaneously. Furthermore, Xiao Tan He Wei Decoction could inhibit nuclear factor kappa-light-chain-enhancer (NF-kB) activity and inhibit its transfer from the cytoplasm to the nucleus. However, when we incubated with NF-κB activator PMA, the effect of Xiao Tan He Wei Decoction was reversed. These results suggested that Xiao Tan He Wei Decoction could be used as a method for the treatment of gastric precancerous lesions, and possibly provide a theoretical basis for the clinical treatment of gastric cancer and gastric precancerous lesions.

Topics: Animals; Apoptosis; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cell Transformation, Neoplastic; Disease Models, Animal; Drugs, Chinese Herbal; Epithelial-Mesenchymal Transition; Humans; Hyperplasia; Methylnitronitrosoguanidine; NF-kappa B; Precancerous Conditions; Rats, Wistar; Stomach Neoplasms; Tetradecanoylphorbol Acetate

Mesenchymal stem cells (MSCs) are a critical component of the tumor microenvironment. Upon distinct pathological stimulus, MSCs show phenotypic and functional changes. Gastric cancer is one of the leading causes of cancer‑related deaths worldwide. The roles and mechanisms of MSCs in gastric cancer have not been well characterized. In the present study, we investigated the roles of MSCs in the malignant transformation from gastritis to gastric cancer using an N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced gastric cancer model. We isolated MSCs from the gastric tissues of normal (RGN-MSCs) and MNNG-exposed rats (RGI-MSCs), and compared the biological properties of RGI-MSCs with RGN-MSCs. We found that RGI-MSCs had increased proliferative and migratory capabilities than these capacities noted in the RGN-MSCs. In addition, RGI-MSCs produced higher levels of IL-6, CXCL10 and MCP-1 than RGN-MSCs. Moreover, RGI-MSCs promoted the migration of normal gastric mucosa epithelial cells by inducing epithelial-mesenchymal transition (EMT). The upregulation of miR-374 in RGI-MSCs was partially responsible for their increased proliferative and migratory capabilities. Collectively, our findings provide new evidence for the roles of MSCs in gastric carcinogenesis, suggesting that targeting gastric cancer-associated MSCs may represent a novel avenue for gastric cancer therapy.

Topics: Animals; Cell Movement; Cell Proliferation; Cell Transformation, Neoplastic; Chemokine CXCL10; Epithelial-Mesenchymal Transition; Gastritis; Humans; Interleukin-6; Mesenchymal Stem Cells; Methylnitronitrosoguanidine; MicroRNAs; Neoplastic Stem Cells; Rats; Stomach Neoplasms; Tumor Microenvironment

Hepatic oval cells are considered as facultative progenitor/stem cells of liver and able to differentiate into either hepatocytes or biliary epithelial cells. The transformed oval cells by carcinogen possess potential to develop carcinomas in animal models. In order to better understand the molecular mechanism in carcinogenetic process, we used a proteomic approach to assess the early changes in protein expression of oval cells (OC3W3-15) initiated by methylnitronitrosoguanidine (MNNG). Meanwhile, we compared cell biologic characteristics of the MNNG treated OC3W3-15 cells and control oval cells by electron microscopy, flow cytometry, karyotype and soft agar assay. The mRNA levels of GGT and GSTP1 determined by real-time PCR were also detected in both cell lines. Our results showed that MNNG-treated OC3W3-15 cells exhibited characteristics of malignant transformation, including growth rate, chromosomal aberrations, abnormal DNA content, and the ability to form colonies. The cells expressed higher levels of the tumor marker AFP, GGT and GSTP1 mRNA than that of control cells. Significant changes of several proteins involved in the malignant transformation process, including cell cycle related proteins, proteins involved in organism development and cell differentiation, are found in OC3W3-15 cells. The proteins may provide early affection in malignant transformation of hepatic oval cells, and yield further insight into mechanism of carcinogenesis of hepatocellular carcinoma.

Topics: Animals; Biomarkers; Cell Culture Techniques; Cell Cycle; Cell Line; Cell Proliferation; Cell Transformation, Neoplastic; Epithelial Cells; Flow Cytometry; Hepatocytes; Immunohistochemistry; Karyotype; Methylnitronitrosoguanidine; Protein Biosynthesis; Rats; Real-Time Polymerase Chain Reaction; RNA, Messenger; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

MicroRNAs (miRNAs) are recently discovered regulators of gene expression and are important in the regulation of many cellular events. Evidence collected to date shows that miRNAs are altered after exposure to environmental toxicants. However, the role that miR-21 plays in the gastric tumorigenesis induced by environmental carcinogens remains largely unknown. The aim of this study was to characterize the regulatory role of miR-21 in the carcinogenic processes following exposure to the N-nitroso carcinogen N-methyl-N-nitro-N'-nitrosoguanidine (MNNG). We found a progressive dose- and time-dependent increase in miR-21 expression following treatment with MNNG. Dysregulated miR-21 affected both cell growth in GES-1 cells and the gastric tumorigenesis induced with MNNG. These data demonstrate the involvement of miR-21 in the malignant transformation and tumorigenesis activated by MNNG. We also established that the Fas ligand (FASLG) and B-cell translocation gene 2 (BTG2), regulated by miR-21, contribute to the transformation induced by MNNG in GES-1 cells. This is the first study to show that miR-21 is involved in chemical carcinogenesis in vivo and in vitro. The regulation by miR-21 of the gastric carcinogenesis induced by MNNG highlights the functional roles of miRNAs in chemical carcinogenesis, and offers a new explanation of the mechanisms underlying chemical carcinogenesis.

Topics: 3' Untranslated Regions; Animals; Apoptosis; Binding Sites; Carcinogens; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Dose-Response Relationship, Drug; Fas Ligand Protein; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Immediate-Early Proteins; Methylnitronitrosoguanidine; Mice; Mice, Inbred BALB C; Mice, Nude; MicroRNAs; RNA Interference; Stomach Neoplasms; Time Factors; Transfection; Tumor Suppressor Proteins

Targeted disruption of STAT3 function has proven to be a useful cancer therapeutic approach by inducing apoptotic cell death. Cucurbitacin is currently under development as a small molecule of STAT3 inhibitor to trigger cell death in many cancers. Here, we systematically studied the molecular mechanisms underlying cucurbitacin-induced cell death, in particular the involvement of autophagy. Treatment with cucurbitacin resulted in non-apoptotic cell death in a caspase-independent manner. Notably, cucurbitacin enhanced excessive conversion of lipidated LC3 (LC3-II) and accumulation of autophagosomes in many cell types. Such autophagy and cell death induced by cucurbitacin were independent of its ability to inhibit STAT3 function, but mainly mediated by enhanced production of mitochondrial-derived reactive oxygen species (ROS), and subsequently activation of extracellular signal-regulated kinase (ERK) and c-jun NH2-terminal kinase (JNK). Interestingly, both the autophagy inhibitor wortmannin and knockdown of Atg5 or Beclin 1 failed to rescue the cells from cucurbitacin-induced cell death, as suppression of autophagy induced the mode of cell death to shift from autophagic cell death to caspase-dependent apoptosis. Thus the present study provides new insights into the molecular mechanisms underlying cucurbitacin-mediated cell death and supports cucurbitacin as a potential anti-cancer drug through modulating the balance between autophagic and apoptotic modes of cell death.

Topics: Apoptosis; Autophagy; Caspases; Cell Line, Tumor; Cell Transformation, Neoplastic; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Humans; JNK Mitogen-Activated Protein Kinases; Methylnitronitrosoguanidine; Mitochondria; Models, Biological; Phenanthridines; Reactive Oxygen Species; STAT3 Transcription Factor; Triterpenes

The SHE cell transformation assay has traditionally been conducted with a feeder layer of X-ray irradiated cells to provide growth support to the target cells seeded in low numbers. The need for an X-ray irradiated feeder cell layer necessitates the maintenance of an X-ray machine and the additional step to seed feeder cells prior to plating target cells. This laboratory has previously reported a method allowing target cells to be seeded in conditioned media prepared from the stock culture flasks in lieu of plating them on a feeder layer (Pant et al. [1,2,4]). In order to expand the data base for chemicals tested using this method, we describe in this paper the results obtained testing Di(2-ethylhexyl)phthalate (DEHP) and N-nitroso-N-methylnitroguanidine (MNNG) which are known to give positive responses in the standard SHE cell transformation assay. With freshly prepared conditioned medium (used within 2 weeks of preparation), there was essentially no difference in the number of target cell colonies in the conditioned medium and in the plates with the X-ray irradiated feeder cell layer. The plating efficiencies of the vehicle controls were within the historical range for the standard SHE cell transformation assay. In more than ten experiments the positive control benzo(a)pyrene [B(a)P] elicited a significant increase in morphological transformation frequency (MTF), with or without X-ray irradiated feeder cells. Compounds, DEHP and MNNG, were tested in the SHE cell transformation assay with and without an X-ray irradiated feeder layer and using a 7-day exposure regimen. The results were comparable between experiments performed using either method. These results demonstrate the feasibility of conducting the SHE cell transformation assay without the use of an X-ray irradiated feeder layer, thereby simplifying the test procedure and assisting the scoring of morphologically transformed colonies.

Topics: Animals; Carcinogenicity Tests; Cell Culture Techniques; Cell Transformation, Neoplastic; Cricetinae; Diethylhexyl Phthalate; Embryo, Mammalian; Mesocricetus; Methylnitronitrosoguanidine

Gastric cancer results from a combination of Helicobacter pylori (H pylori) infection, exposure to dietary carcinogens, and predisposing genetic make-up. Because the role of these factors in gastric carcinogenesis cannot be determined readily in human beings, the present study examined the role of an oral carcinogen and H pylori infection in rhesus monkeys.. Gastroscopies were performed in 23 monkeys assigned to 4 groups: controls; nitrosating carcinogen ethyl-nitro-nitrosoguanidine administration alone; inoculation of a virulent H pylori strain alone (H); and ethyl-nitro-nitrosoguanidine in combination with H pylori (EH). Follow-up gastroscopies and biopsies were performed at 3-month intervals for 5 years for pathologic and molecular studies.. Postinoculation, H and EH groups showed persistent infection and antral gastritis. Starting at 2 and 5 years, respectively, gastric intestinal metaplasia and intraepithelial neoplasia developed in 3 EH monkeys but in no other groups. Transcriptional analysis of biopsy specimens at 5 years revealed group-specific expression profiles, with striking changes in EH monkeys, plus a neoplasia-specific expression profile characterized by changes in multiple cancer-associated genes. Importantly, this neoplastic profile was evident in nonneoplastic mucosa, suggesting that the identified genes may represent markers preceding cancer.. Gastric intraglandular neoplasia is induced in primates when H pylori infection is associated with consumption of a carcinogen similar to the nitrosamines found in pickled vegetables, suggesting that H pylori and the carcinogen synergistically induce gastric neoplasia in primates.

Topics: Animals; Biopsy; Carcinogens; Carcinoma in Situ; Cell Transformation, Neoplastic; Cluster Analysis; Diet; Disease Models, Animal; Disease Progression; DNA Repair; Female; Gastritis; Gastroscopy; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Helicobacter Infections; Helicobacter pylori; Macaca mulatta; Male; Metaplasia; Methylnitronitrosoguanidine; Oligonucleotide Array Sequence Analysis; Precancerous Conditions; Stomach Neoplasms; Time Factors

To investigate the effects of Radix notoginseng extracts drug-containing serum on the expressions of apoptosis-regulating proteins including Bax, bcl-2 and p21WAF1 in precancerous gastric cells.. The N-methyl-N'-nitro-N-nitroso-guanidine (MNNG) transformed eternalized human gastric mucosa epithelium GES-1 cell line (MC cell) was used in vitro as a model of gastric precancerous lesion. The medicated canine serum was prepared by feeding to the adult Beagle dog with Radix notoginseng extracts and obtaining the serum after 2-hour medication. MC cells were cultured with medicated canine serum (medicated serum group) or non-medicated canine serum (normal control group) for 72 hours. Expressions of Bax, bcl-2 and p21WAF1 proteins were detected by immunocytochemical assay and the average optical density of the cells was determined by an image analysis system.. Compared with those of the normal control group, Bax and p21WAF1 expressions in medicated serum group were significantly enhanced (P<0.01), while the expression of bcl-2 was significantly reduced (P<0.01).. Radix notoginseng extracts may inhibit the proliferation and promote the apoptosis of precancerous gastric cells through altering expressions of the bcl-2, Bax and p21WAF1 genes.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin-Dependent Kinase Inhibitor p21; Dogs; Drugs, Chinese Herbal; Gastric Mucosa; Humans; Methylnitronitrosoguanidine; Panax notoginseng; Plant Roots; Precancerous Conditions; Proto-Oncogene Proteins c-bcl-2; Serum; Stomach Neoplasms

To investigate the role and possible mechanism of JWA in N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) inducing human bronchial epithelial (HBE) cells' neoplastic transformation.. JWA overexpression vector and its stable transfection HBE cells were established. The characteristics of transformed HBE cells were determined by methyl thiazolyl tetrazolium (MTT) assay and the soft agar colony formation assay. The expressions of JWA and P53 were detected by Western blot.. The growth rates of the HBE cells which were treated with MNNG were significantly accelerated than the JWA overexpression HBE cells and controlled HBE cells (P < 0.05). The soft agar colony formation of JWA overexpression HBE cells with and without MNNG treatment (8.06% and 10.14%) was significantly lower than that of the normal HBE cells with MNNG treatment (26.80%) (P < 0.01). After exposure of MNNG, the P53 expressions were gradually increased in HBE cells with the increased passages. However, the expression of P53 in JWA over expressed HBE cells showed a different manner. P53 reached an over expression peak at early stage (the first passage), and then with a gradually down-regulated expression spectrum with increased passages of the cells.. JWA might be a key molecule and play an important role in MNNG inducing neoplastic transformation in HBE cells through regulation of the expression of P53.

Topics: Cell Transformation, Neoplastic; Cells, Cultured; Epithelial Cells; Heat-Shock Proteins; Humans; Intracellular Signaling Peptides and Proteins; Membrane Transport Proteins; Methylnitronitrosoguanidine; Tumor Suppressor Protein p53

Okadaic acid (OA) is a tumor promoter in two-stage carcinogenesis experiments. Nevertheless, the effects of OA on cell transformation, cell proliferation and apoptosis vary widely, and the molecular events underlying these effects of OA are not well understood. In the present study, we examined the promoting activity and the associated effects on cell growth and apoptosis mediated by OA in BALB/c 3T3 cells, and evaluated alterations of gene transcriptional expression by microarray analysis. The promoting activity of OA was estimated by a two-stage transformation assay, in which cells were treated first with a low dose of the initiator N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and then with OA for 14 days. It showed that OA, at concentrations of 7.8-31.3 ng/ml, enhanced the transformation of MNNG-treated cells. In the promotion phase, cells exposed to OA (7.8 ng/ml) grew slowly for the first 2 days and subsequently died. As determined by Hoechst 33342 fluorescent dye and Annexin-V/PI dual-colored flow cytometry, OA induced morphologically apoptotic cells and increased the percentage of early apoptotic cells. The gene expression profile induced by OA at five time points in the promotion phase was determined by use of a specific mouse toxicological microarray containing 1796 clones, and a total of 177 differentially expressed genes were identified. By gene ontology analysis, 31 of these were determined to be functionally involved with cell growth and/or maintenance. In this group, numerous genes associated with the cell proliferation and cell cycle progression were down-regulated at early and/or middle time points. Among these was a subset of genes associated with apoptosis, in which Bnip3, Cycs, Casp3 and Bag1 genes are involved in the mitochondrial pathway of apoptosis. Ier3, Mdm2 and Bnip3 genes may be p53 targets. Furthermore, real-time PCR confirmed the expression changes of five genes selected at random from the differentially expressed genes. We conclude that OA induces cell growth inhibition and apoptosis in the two-stage, MNNG-initiated transformation of BALB/c 3T3 cells. The results of gene expression profile analysis imply that multiple molecular pathways are involved in OA-induced proliferation inhibition and apoptosis. Mitochondrial and p53-associated apoptotic pathways also may contribute to OA-induced apoptosis.

Topics: 3T3 Cells; Animals; Apoptosis; Carcinogens; Cell Cycle; Cell Proliferation; Cell Transformation, Neoplastic; Dose-Response Relationship, Drug; Gene Expression Regulation; Methylnitronitrosoguanidine; Mice; Mice, Inbred BALB C; Mitochondria; Okadaic Acid; Oligonucleotide Array Sequence Analysis; Time Factors; Tumor Suppressor Protein p53

Genomic imprinting, a heritable form of epigenetic information, is thought to play an important role in tumor progression. DNA methylation is a common mechanism of genomic imprinting. To evaluate the genome-wide effects of malignant transformation on osteosarcoma progression, we examined multiple biological properties, including DNA methylation, in human osteoblast hFOB1.19 cells (ATCC Catalog No. CRL-11372) transformed by treatment with carcinogenic agent N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG, 1.0 microg/ml) and carcinogenic promoting agent 12-O-tetradecanoyl phorbol-13-acetate (TPA, 200 ng/ml). We also examined global changes in expression of imprinted genes during transformation using microarray analysis. Ten imprinted genes, including H19, MKRN3, NDN, CDKN1C, PHLDA2, MEST, CD81, GRB10, SLC22A18, and SLC22A3 were aberrantly regulated in transformed cells, suggesting roles in tumorigenesis. Moreover, we analyzed the methylation state of the promoter regions of H19, PHLDA2, and SLC22A18 genes by bisulfite sequencing array and observed a correlation between upregulated expression of H19 and PHLDA2 genes and hypomethylation of their promoter regions, although this was not observed for SLC22A18. Our results suggest that changes in expression of imprinted genes caused by changes in methylation are involved, and are among the earliest events, in neoplastic progression.

Topics: Bone Neoplasms; Carcinogens; Cell Line; Cell Transformation, Neoplastic; DNA Methylation; Gene Expression Regulation, Neoplastic; Genomic Imprinting; Humans; Methylnitronitrosoguanidine; Oligonucleotide Array Sequence Analysis; Osteoblasts; Osteosarcoma; Tetradecanoylphorbol Acetate

Heat shock factor 1 (HSF1) is the master regulator of the heat shock response in eukaryotes, a very highly conserved protective mechanism. HSF1 function increases survival under a great many pathophysiological conditions. How it might be involved in malignancy remains largely unexplored. We report that eliminating HSF1 protects mice from tumors induced by mutations of the RAS oncogene or a hot spot mutation in the tumor suppressor p53. In cell culture, HSF1 supports malignant transformation by orchestrating a network of core cellular functions including proliferation, survival, protein synthesis, and glucose metabolism. The striking effects of HSF1 on oncogenic transformation are not limited to mouse systems or tumor initiation; human cancer lines of diverse origins show much greater dependence on HSF1 function to maintain proliferation and survival than their nontransformed counterparts. While it enhances organismal survival and longevity under most circumstances, HSF1 has the opposite effect in supporting the lethal phenomenon of cancer.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinogens; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cell Transformation, Neoplastic; DNA-Binding Proteins; Fibroblasts; Gene Expression Regulation, Neoplastic; Genotype; Glucose; Heat Shock Transcription Factors; Humans; Methylnitronitrosoguanidine; Mice; Mice, Inbred BALB C; Mice, Knockout; Mutation; Phenotype; Protein Biosynthesis; Proto-Oncogene Proteins c-sis; ras Proteins; RNA Interference; RNA, Small Interfering; Signal Transduction; Skin; Skin Neoplasms; Tetradecanoylphorbol Acetate; Time Factors; Transcription Factors; Transduction, Genetic; Tumor Suppressor Protein p53

To obtain new Se resources with high healthy value (both high activity and low toxicity), the preventive efficacies of three Se-enriched higher plants on stomach cancer were compared with selenite and Se-enriched garlic.. Ninety weanling male Wistar rats were fed the basal diet for a week, divided equally into nine groups, control, MNNG,Se 75 and 150 microg/kg bw of selenite, Se 150 and 300 microg/kg bw of Se-enriched garlic, Se 150 microg/kg bw of Se-enriched broccoli, Se 300 microg/kg bw of Se-enriched red kales and green kales group. Rats in MNNG and Se supplementation groups were daily given 15 mg/kg bw of MNNG (solved in 1 ml distilled water) and the rats of control group were given 1 ml distilled water by gavage for ten days. Meanwhile, the rats of the control and MNNG group were daily given 1 ml distilled water and the rats of other groups were given 1 ml water suspension of Se-enriched garlic, red kavas, green kavas, broccoli or 1 ml solution of sodium selenite by gavage for seventeen weeks. All rats were freely fed the basal diet and water during the period of the experiment. At the end of 18th week, the rats were sacrificed, the incidence of aneuploid cells (IAC) in mucosal epithelium of the gastric antrum was detected, and the IAC data were analyzed by SPSS 12.0.. The results showed that the IACs were 25% and 30%, respectively, in the Se 150 microg/kg bw of Se-enriched garlic and -broccoli group, and were in turn 22%, 50% and 30% in the 300 microg/kg bw of Se-enriched garlic, -red kale and -green kale. No significant differences of IACs were found in the same level of Se supplementation groups by Se-enriched plants.. The data showed that Se-enriched broccoli, red kale and green kale had high activities similar to Se-enriched garlic in stomach cancer prevention and lower toxicity than selenite.

Topics: Aneuploidy; Animals; Brassica; Cell Transformation, Neoplastic; Cells, Cultured; Epithelial Cells; Garlic; Gastric Mucosa; Male; Methylnitronitrosoguanidine; Pyloric Antrum; Rats; Rats, Wistar; Selenium; Stomach Neoplasms

The transforming effect of N-methyl-N-nitro-N-nitrosoguanidine (MNNG) on cultured cells from Schistosoma japonicum (S. japonicum) was studied using mono-factor and orthogonal tests. Under the influence of MNNG, cultured cells grew well, and cell survival time was more than 246 days in low-serum medium. When treated with 3 mug/ml MNNG for 48 h, the number of dividing cells increased significantly as determined by bright-field and scanning electron microscopy (SEM). Under these conditions, abundant microvilli, ruffles, microridges, papillae and blebs were observed on the surface of the induced cells. Treatment with MNNG may overcome existing limitations to get continually proliferating schistosome cells and open the possibility to immortalize isolated cells.

Topics: Animals; Cell Survival; Cell Transformation, Neoplastic; Cells, Cultured; Dose-Response Relationship, Drug; Methylnitronitrosoguanidine; Microscopy; Microscopy, Electron, Scanning; Microvilli; Mutagens; Schistosoma japonicum

To elucidate the potential molecular mechanism responsible for the early time of tumor promotion, gene expression profile was studied in the transformed BALB/c 3T3 cells induced by 12-O-tetradecanoylphorbol-13-acetate (TPA).. The two-stage cell transformation model was established by using the initiator of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and promoter of TPA. Cell proliferation was measured by trypan blue staining and cell cycle analysis was carried out by flow cytometry assay. A cDNA microarray representing 1 152 genes was used to investigate the gene expression profiles of BALB/c 3T3 cells exposed to TPA at 4 h and 24 h respectively.. TPA could effectively inhibit cell proliferation and induce the G1 and S cell cycle arrested in the early time. Moreover 19 genes were found differentially expressed at least twofold in the TPA treated cells as compared with the control cells, 9 of them were upregulated and 10 downregulated. Most of the differentially expressed genes were involved in cell proliferation, differentiation or apoptosis, and related to ras or p53 signal transduction pathway.. TPA could influence the transcriptional expression of some genes related to cell cycle modulation and ultimately result in the cell growth arrest.

Topics: Animals; Apoptosis; BALB 3T3 Cells; Cell Cycle; Cell Differentiation; Cell Proliferation; Cell Transformation, Neoplastic; Flow Cytometry; Gene Expression; Gene Expression Profiling; Methylnitronitrosoguanidine; Mice; Oligonucleotide Array Sequence Analysis; Tetradecanoylphorbol Acetate

Global genomic changes, including DNA aneuploidy, may be necessary for carcinogenesis; however, such genomic changes in precancerous cells have not been studied extensively. To identify early global genotypic changes associated with precancerous lesions, a non-transformed human liver epithelial cell line, THLE-3, was treated with benzo[a]pyrene or N-methyl-N-nitro-N-nitrosoguanidine, then by 12-O-tetradecanoyl-phorbol-13-acetate, resulting in morphological transformation of cells. We examined genotypic changes of the transformed cells by laser scanning cytometry, fluorescence in situ hybridization, and comparative genomic hybridization. Transformed fusiform cells displayed tetraploidy, chromosomal instability, DNA copy number aberrations. Cells with these changes were still in the precancerous stage. However, it is suggested that these global genomic changes including tetraploidization provide cells with genetic alterations leading to cancer.

Topics: Benzo(a)pyrene; Carcinogens; Cell Line; Cell Transformation, Neoplastic; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 17; DNA; DNA Damage; G1 Phase; Gene Expression Regulation, Neoplastic; Genome, Human; Genotype; Humans; In Situ Hybridization, Fluorescence; Laser Scanning Cytometry; Liver; Methylnitronitrosoguanidine; Neoplasms; Nucleic Acid Hybridization; Phenotype; Ploidies; Resting Phase, Cell Cycle; Tetradecanoylphorbol Acetate; Time Factors

To study the effect drug contained canine serum, prepared by gastric perfusion with Sanchi extract (SE), in inhibiting proliferation and promoting apoptosis of cultured precancerous gastric cells by cell culture.. The precancerous model cells (MC) used in the experiment were prepared through transforming eternalized human gastric mucosa epithelial cells GES-1 by N-methyl-N'-nitro-N-nitroso-guanidine (MNNG). After once gastric perfusion of SE extract to dogs, the canine serum gotten before and at different time points after medication was used for test. The inhibitory effect of the drug serum obtained at different time points on MC after acting for 72 hrs was detected by 3-(4,5)-dimethy thioazol-2-yl-2,5-diphenyl-tetrazoliumbromide (MTT) method to find the optimal time point for drug serum preparation, that were 2 hrs and 6 hrs after medication. Then the cell apoptosis promoting effect after acting for 72 hrs of the drug serum obtained at the optimal time points was determined by flow cytometry.. The drug serum obtained at 2-hr and 6-hr after medication showed the highest inhibitive effect on MC cells, reaching 45.3% and 42.4% respectively, as compared with the effect of blank serum, the difference was significant (P<0.01). They could evidently promote the MC cell apoptosis, the apoptosis rate also showed significant difference to that of the blank serum (P < 0.05). Under their action, the proportion of MC cells in G0/G1 phase was obviously decreased (P < 0.05) while that in the G2/M phase significantly increased (P <0.05). However, the change of cells in S phase was not uniform.. The drug contained canine serum gotten 2 hr and 6 hr after SE feeding shows the optimal MC proliferation inhibitive effect and significant apoptosis promoting effect. Besides, it could significantly decrease the proportion of MC cells in G0/G1 phase and significantly increase that in G2/M phase, this effect might be one of the mechanisms of ES in inhibiting MC cell proliferation and promoting its apoptosis.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Araliaceae; Cell Proliferation; Cell Transformation, Neoplastic; Cells, Cultured; Dogs; Drugs, Chinese Herbal; Embryo, Mammalian; Gastric Mucosa; Ginsenosides; Humans; Methylnitronitrosoguanidine; Precancerous Conditions; Stomach Neoplasms

The tumor suppressor BRCA1 contains multiple functional domains that interact with many proteins. After DNA damage, BRCA1 is phosphorylated by CHK2 at serine 988, followed by a change in its intracellular location. To study the functions of CHK2-dependent phosphorylation of BRCA1, we generated a mouse model carrying the mutation S971A (S971 in mouse Brca1 corresponds to S988 in human BRCA1) by gene targeting. Brca1(S971A/S971A) mice were born at the expected ratio without a developmental defect, unlike previously reported Brca1 mutant mice. However, Brca1(S971A/S971A) mice suffered a moderately increased risk of spontaneous tumor formation, with a majority of females developing uterus hyperplasia and ovarian abnormalities by 2 years of age. After treatment with DNA-damaging agents, Brca1(S971A/S971A) mice exhibited several abnormalities, including increased body weight, abnormal hair growth pattern, lymphoma, mammary tumors, and endometrial tumors. In addition, the onset of tumor formation became accelerated, and 80% of the mutant mice had developed tumors by 1 year of age. We demonstrated that the Brca1(S971A/S971A) cells displayed reduced ability to activate the G(2)/M cell cycle checkpoint upon gamma-irradiation and to stabilize p53 following N-methyl-N'-nitro-N-nitrosoguanidine treatment. These observations suggest that Chk2 phosphorylation of S971 is involved in Brca1 function in modulating the DNA damage response and repressing tumor formation.

Topics: Aging; Animals; BRCA1 Protein; Carcinogens; Cell Cycle; Cell Transformation, Neoplastic; Checkpoint Kinase 2; DNA Damage; Female; Gamma Rays; Hyperplasia; Mammary Glands, Animal; Methylnitronitrosoguanidine; Mice; Mutagenesis, Site-Directed; Mutation; Phenotype; Phosphorylation; Protein Serine-Threonine Kinases; Serine; Survival Rate; Uterus

Through cell cultivation, we studied the inhibiting effects of the serum of the dog fed with Panax notoginseng extracts on precancerous gastric cells, trying to find the best time points or periods when the extracts' function was the strongest after administration of the extracts to the dog.. The experiments adopted eternalized human gastric mucosa epithelium GES-1 cells and MC cells gained from GES-1 cells transformed by N-methyl-N'-nitro-N-nitroso-guanidine (MNNG) as the model of precancerous lesions for study in vitro. We took the serum of a dog before and at different points of time after feeding the dog with Panax notoginseng extracts for experiment. By means of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay, we examined the inhibiting effects of the serum after culturing the GES-1 and MC cells for 72 hours with different concentration (8%, 4%, 2%) of medicated serum obtained from the dog at different points of time, so as to find that, at which points of time the medicated serum obtained, it would be the most effective.. The results showed that the GES-1 and MC cells inhibition rates of medicated serum from the points of 2-hour and 6-hour were the highest, and the culture medium containing 8% of medicated serum from these two points had prominent inhibiting effects on both kinds of cells. The GES-1 cells inhibition rate in culture medium containing 8% of medicated serum from the point of 2-hour was 70.8% (P<0.01) and that of the MC cells was 45.3% (P<0.01). The GES-1 cells inhibition rate in culture medium containing 8% of medicated serum from the point of 6-hour was 88.5%(P<0.01) and that of the MC cells was 42.4% (P<0.01).. The points of time with the strongest inhibiting effects are 2 hours and 6 hours after being fed with Panax notoginseng extracts. At these two points, the serum is most effective in inhibiting the proliferation of GES-1 and MC cells.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Dogs; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; Immune Sera; Male; Methylnitronitrosoguanidine; Panax; Plant Extracts; Stomach Neoplasms; Time Factors

The fact that certain ultraviolet (UV) filters used in cosmetics display estrogenic activity prompted us to study potential actions on androgen receptors (AR) in the human breast carcinoma cell line MDA-kb2, which expresses functional endogenous AR and glucocorticoid receptors (GR) and is stably transfected with a luciferase reporter plasmid. Dihydrotestosterone (DHT), methyltrienolone (R1881), methyltestosterone, danazol, and androstenedione increased luciferase activity, with EC50 values between 0.11 nM (R1881), 0.14 nM (DHT), and 73.5 nM (androstenedione). DHT-induced luciferase gene expression was inhibited by nonsteroidal antiandrogens, hydroxyflutamide, flutamide, bicalutamide, and vinclozolin. In contrast, the steroidal AR agonist/antagonist cyproterone actetate showed agonistic activity in the absence and presence of DHT, which was not blocked by hydroxyflutamide and thus seems not to be mediated by AR. GR-mediated activation of luciferase by dexamethasone was 100 times less potent than DHT and was not antagonized by hydroxyflutamide. The cell line was used for screening of UV filters, benzophenone-3 (Bp-3), benzophenone-4, 3-benzylidene camphor, 4-methylbenzylidene camphor, butyl-methoxy-dibenzoylmethane, homosalate (HMS), octyl-dimethyl-PABA, and octyl-methoxycinnamate. Two of these, Bp-3 and HMS, antagonized DHT-induced AR activation below cytotoxic concentrations, with IC50 of 5.57 10-6 M (HMS) and 4.98 10-6 M (Bp-3). None of the eight UV filters displayed agonistic activity when tested alone, but high concentrations of Bp-3 induced an increase of luciferase activity in the presence of dexamethasone, which was not blocked by hydroxyflutamide or the estrogen antagonist, ICI 182,780. These data indicate that the UV filters Bp-3 and HMS possess antiandrogenic activity in vitro in addition to estrogenic activity.

Topics: Arsenic; Biomarkers; Carcinogens; Cell Line; Cell Transformation, Neoplastic; DNA Primers; Gene Expression Regulation, Neoplastic; Humans; Keratinocytes; Metals; Methylnitronitrosoguanidine; Oligonucleotide Array Sequence Analysis; Reverse Transcriptase Polymerase Chain Reaction

The enhancement of carcinogen-induced malignant transformation of C3H/M2 mouse fibroblasts by the tumor promoters 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 12-O-tetradecanoylphorbol-13-acetate (TPA) is associated with the induction of cyclooxygenase expression and the stimulation of prostaglandin (PG) formation. Therefore, the potential of PGs, i.e., PGF(2alpha) and PGE(2), for tumor promotion was studied in the two-step C3H/M2 cell transformation assay, a model of the multi-step process of carcinogenesis. The transformation of fibroblasts was clearly enhanced by the addition of PGF(2alpha) in the promotion phase after pretreatment with a subthreshold dose of a carcinogen (3-methylcholanthrene or N-methyl-N'-nitro-N-nitrosoguanidine). No enhancement of cell transformation was observed in cells without carcinogen-pretreatment, i.e., PGF(2alpha) had no tumor initiating potential. The promotional effect was dose-dependent with a maximum at 16 nM PGF(2alpha). PGE(2) had no significant effect in this assay. Furthermore, PGF(2alpha) (but not PGE(2)) clearly reduced the inhibition of TPA-induced promotion by NS-398, an isozyme-specific inhibitor of cyclooxygenase-2. The inhibition of TPA- or TCDD-induced promotion by the non-specific cyclooxygenase inhibitor indomethacin was not affected by co-treatment with PGF(2alpha) and PGE(2). Our data suggest that PGF(2alpha) acts as an endogenous promoter of cell transformation implying that it may also be critically involved in tumor promoter-induced signalling transfer cascades ultimately triggering the process of carcinogenesis.

Topics: Animals; Carcinogens; Cell Transformation, Neoplastic; Cyclooxygenase Inhibitors; Dinoprost; Dinoprostone; Dose-Response Relationship, Drug; Drug Synergism; Fibroblasts; Methylcholanthrene; Methylnitronitrosoguanidine; Mice; Mice, Inbred C3H; Nitrobenzenes; Polychlorinated Dibenzodioxins; Sulfonamides; Tetradecanoylphorbol Acetate

Cadmium (Cd), a carcinogenic metal in human and rodents, has been shown to transform cells in vitro. However, the carcinogenic mechanisms of Cd as a mutagen and/or promoter are not well clarified. We already reported that CdCl2 in a range of 1.5 approximately 360 ng/ml enhanced transformation of Balb/3T3 A31 cells induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG, 0.1 microg/ml) in a dose-dependent manner (Fang et al., Toxicol. In Vitro 15(3) (2001a) 51-7). In previous study, we observed that Cd stimulated cell proliferation on MNNG-initiated cells through inactivation of p53 and p27 and increase of proliferating cell nuclear antigen (PCNA) expression after 24 h treatment (Fang et al., Toxicology 163 (2001b) 175-84). The aim of this study is to further elucidate the long-term effect of Cd in terms of cell cycle control gene expressions during the promotion stage of in vitro two-stage transformation. For the purpose, we determined the expression levels of the genes involved in growth regulation, such as p53, p27, c-myc, mdm2, cyclins D1 and B1, CDK4, and PCNA in the cells treated with Cd for 14 days after MNNG-initiation. In MNNG+CdCl2 group, cells apparently expressed cellular tumor antigen p53 mRNA, but did not express the wild-type p53 protein; the protein and mRNA levels of p27 were reduced apparently in the cells of MNNG+CdCl2 group compared to the cells of control and MNNG group. In addition, the protein levels of cyclin D1, CDK4, PCNA, c-myc, and mdm2, and cyclin B1 mRNA level were higher in MNNG+CdCl2 group than control and MNNG group. Together with previous data (Fang et al., Toxicology 163 (2001b) 175-84), our results indicated that during the transformation process of MNNG-treated cells, Cd may activate oncogenes such as c-myc, mdm2, and cellular tumor antigen p53, inhibit the tumor suppressor genes such as wild-type p53 and p27, and consequently accelerate the proliferation of initiated cells. This work firstly demonstrates that Cd affects the genes involved in growth regulation on initiated cells during the promotion stage of in vitro cell transformation.

Topics: 3T3 Cells; Animals; Cadmium; Cell Division; Cell Transformation, Neoplastic; DNA Damage; Gene Expression; Genes, myc; Methylnitronitrosoguanidine; Mice; Microfilament Proteins; Muscle Proteins; Nuclear Proteins; Proliferating Cell Nuclear Antigen; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-mdm2; RNA, Messenger; Tumor Suppressor Protein p53

The mucosal changes by which duodenogastric reflux may predispose to gastric cancer have not been fully clarified. In this study in rats, duodenal fluid was directed into the stomach through a gastroenterostomy (jejunal reflux, N = 29) or through the pylorus (pyloric reflux, N = 30) and compared with 30 controls. Twenty-four weeks later the stomach was exposed to N-[3H]methyl-N-nitro-N-nitrosoguanidine ([3H]MNNG). The corpus mucosa was examined for proliferating cells (bromodeoxyuridine labeled) and cells at risk of methyl-N-nitro-N-nitrosoguanidine-induced carcinogenesis (cells labeled with bromodeoxyuridine and [3H]MNNG). The number of double-labeled cells increased from 0.8 +/- 0.1/mm mucosa in the control group to 5.2 +/- 0.9 in the jejunal reflux group (P < 0.05) and 2.7 +/- 0.5 in the pyloric reflux group (P < 0.05). An erosion or ulcer appeared at the gastroenterostomy in 52% of animals with jejunal reflux and 17% of those with pyloric reflux (P < 0.006). Within erosions the mean number of double-labeled cells was 9.6 +/- 2.2 in the jejunal reflux group and 7.7 +/- 4.8 in the pyloric reflux group, and significantly higher than in the nonlesion area of the mucosa (0.6 +/- 0.2 and 0.8 +/- 0.3). In erosions the distance between the gastric lumen and the proliferating cells was significantly shorter and the cell proliferation significantly higher than in the nonlesion area of the mucosa. We conclude that duodenogastric reflux increases the penetration of [3H]MNNG into the corpus mucosa of rats and also induces mucosa lesions, which further increase the penetration of [3H]MNNG into the corpus mucosa.

Topics: Animals; Cell Transformation, Neoplastic; Duodenogastric Reflux; Gastric Mucosa; Male; Methylnitronitrosoguanidine; Rats; Rats, Wistar; Stomach Neoplasms; Tritium

The identification of molecular markers related to critical biological processes during carcinogenesis may aid in the evaluation of carcinogenic potentials of chemicals and chemical mixtures. Work from our laboratory demonstrated that a single treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) enhanced spontaneous malignant transformation of the human keratinocyte cell line RHEK-1. In contrast, chronic low-level exposure of cells to arsenic alone or in a mixture containing arsenic, cadmium, chromium, and lead inhibited malignant conversion. To identify changes in gene expression that influence these different outcomes, cDNA microarray technology was used. Analysis of multiple human arrays in MNNG-transformed RHEK-1 cells, designated OM3, and those treated with arsenic or the arsenic-containing metal mixture showed unique patterns of gene expression. Genes that were overexpressed in OM3 included oncogenes, cell cycle regulators, and those involved in signal transduction, whereas genes for DNA repair enzymes and inhibitors of transformation and metastasis were suppressed. In arsenic-treated cells, multiple DNA repair proteins were overexpressed. Mixture-treated cells showed increased expression of a variety of genes including metallothioneins and integrin 4. These cells showed decreased expression of oncogenes, DNA repair proteins, and genes involved in the mitogen-activated protein kinase pathway. For comparison we are currently analyzing gene expression changes in RHEK-1 cells transformed by other means. The goal of these studies is to identify common batteries of genes affected by chemical modulators of the carcinogenic process. Mechanistic studies may allow us to correlate alterations in their expression with sequential stages in the carcinogenic process and may aid in the risk assessment of other xenobiotics.

Topics: Arsenic; Cell Culture Techniques; Cell Transformation, Neoplastic; DNA Repair; Gene Expression Profiling; Gene Expression Regulation; Genetic Markers; Humans; Keratinocytes; Methylnitronitrosoguanidine; Oligonucleotide Array Sequence Analysis; Protein Kinases; Risk Assessment; Xenobiotics

At present, the effect of cyclin D2 implicated in cell cycle regulation, differentiation, and oncogenic transformation is not fully confirmed. To better elucidate the role of cyclin D2 in controling the cell proliferation, cyclin D2 expression level was determined at the early initiation and promotion stages during the in vitro two-stage transformation process of Balb/3T3 A31 cells. N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced G2/M-arrested cells expressed low level of cyclin D2 mRNA, while the contact-inhibited nonproliferating cells expressed high level of cyclin D2 mRNA. In the transformed proliferating cells at the promotion stage, cyclin D2 mRNA was not expressed. These data suggest that cyclin D2 expression may be associated with the type of growth arrest and nonproliferating state, but not with the cell proliferation and transformation.

Topics: 3T3 Cells; Animals; Blotting, Northern; Cell Division; Cell Transformation, Neoplastic; Cyclin D2; Cyclins; G2 Phase; Methylnitronitrosoguanidine; Mice; Mice, Inbred BALB C; Mitosis; Proliferating Cell Nuclear Antigen; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

During the multistage carcinogenesis, functions of several key genes involved in the cell cycle control and cell-cell communication can be damaged. Gap junction intercellular communication (GJIC) is known to transfer small, water-soluble molecules through intercellular channels composed of proteins called connexins (Cxs). Therefore, aberrant expression of Cx may be one of the critical factors for the clonal expansion of initiated cells during the two-stage transformation. We already improved the classical in vitro two-stage transformation method using N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) as an initiator and cadmium as a promoter on Balb/3T3 A31 cells, and reconfirmed the promotional effect of cadmium with this method (Fang, M.Z., Cho, M.H., Lee, H.W., 2001. Improvement of in vitro two-stage transformation assay and detection of the promotional effect of cadmium, Toxicol. In Vitro (in press). In this study, precise roles of Cd on Cx expression in normal Balb/3T3 A31 and during the promotion stage of the in vitro two-stage transformation were elucidated. For this purpose, the Cx43, Cx32 and Cx26 protein levels, Cx43 and Cx26 mRNA levels and the cellular distribution location of Cx43 protein were determined. Normal Balb/3T3 cells expressed Cx43 and Cx32, but not Cx26. After a short-term treatment of cadmium on normal cells, phosphorylation of Cx43 protein increased and Cx32 protein level decreased. However, during the promotion stage of the in vitro two-stage transformation, transformed cells treated with cadmium for long periods expressed Cx43 and Cx32 highly, similar to the level of normal Balb/3T3 cells, compared to the nontransformed cells. Moreover, Cx43 of the transformed cells was distributed mostly in the perinuclear region rather than the intercellular membrane. These data suggest that cadmium may inhibit the GJIC by increasing the phosphorylation of Cx43 and decreasing the expression of Cx32 in the normal Balb/3T3 A31 cells. Our results also suggest that these changes are not associated with the cell transformation; transformed cells may reexpress Cx43 and Cx32 similar to the normal cells, though Cx43 protein is distributed aberrantly during the transformation process. Further studies are needed to clarify the relationship between transformation and posttranslational modification of the Cx proteins.

Topics: Animals; Blotting, Western; Cadmium; Cell Transformation, Neoplastic; Cells, Cultured; Connexins; Gap Junctions; Methylnitronitrosoguanidine; Mice; Mice, Inbred BALB C

The classical in vitro two-stage transformation method was modified for high transformation frequency, and the promotional effect of cadmium was evaluated. In this study, we reconfirmed the usefulness of the replating method and the optimal duration time between the initiator and promoter treatments for the optimal transformation of the Balb/3T3 cells. The results also showed that subsequent exposure to CdCl(2) for 2 weeks after initial exposure to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) markedly enhanced the transformation frequency. At the concentration of 360 ng/ml, the transformation frequency was increased by 35-fold that of the cultures treated only with MNNG, and was higher than that of the positive control group treated with 100 ng/ml 12-O-tetradecanoyl phorbol-13-acetate (TPA) after MNNG treatment. This transformation frequency was higher than that reported previously. Therefore, this in vitro two-stage transformation method can be used efficiently for the screening of genotoxic and non-genotoxic carcinogens and the study of multistage carcinogenesis. These results also indicate that cadmium has a strong potency as a promoter, and the promotional effect of cadmium is higher than that of TPA.

Topics: 3T3 Cells; Animals; Cadmium; Carcinogenicity Tests; Carcinogens; Cell Line; Cell Transformation, Neoplastic; Dose-Response Relationship, Drug; In Vitro Techniques; Methylnitronitrosoguanidine; Mice; Mice, Inbred BALB C; Polychlorinated Dibenzodioxins; Tetradecanoylphorbol Acetate; Time Factors

In vitro two-stage transformation, an important method for the screening of carcinogens, is a valuable approach for the mechanistic study of multi-stage carcinogenesis. However, very little is known about the molecular and cellular mechanisms, particularly in terms of cell cycle control during in vitro two-stage transformation. We improved the in vitro two-stage transformation method using N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) as an initiator and cadmium as a promoter, and reconfirmed the promotional effect of cadmium (Fang et al., 2001a). To determine the alterations of cell cycle control in the MNNG-induced initiation stage during transformation, we examined the effects of MNNG on Balb/3T3 A31 cell growth, and determined the alterations of the protein and/or mRNA levels of cyclins B1, D1, E, and G, PCNA, GADD45, p27, and wild-type p53. After 4 hour treatment of MNNG, populations of G2/M phase distribution and apoptotic fraction and the cyclin G mRNA level increased, while the cyclin B1 mRNA level decreased in a concentration-dependent manner. Wild-type p53, p27, and GADD45 protein levels also increased as a function of MNNG concentrations. However, cyclin D1, cyclin E, and PCNA expressions remained unchanged. During the initiation stage, PCNA protein expression decreased on the first day after MNNG-treatment, then increased gradually during the following 6 days, and further increased on the first day after cadmium treatment. Although wild-type p53 and p27 protein expressions also showed temporary retardation on the first day after MNNG-treatment, the expressions increased gradually during the following 6 days, but decreased rapidly by the cadmium treatment. These results indicated that during the initiation stage, MNNG induced G2/M arrest and apoptosis with increased expressions of wild-type p53, p27, and GADD45 proteins; and down-regulated mRNA level of cyclin B1 and up-regulated mRNA level of cyclin G. In addition, although a few of the G2/M-arrested cells proliferated gradually, most cells continued to be suppressed and inactivated by the over-expressions of wild-type p53 and p27 until the cadmium treatment.

Topics: 3T3 Cells; Animals; Apoptosis; Carcinogens; Cell Cycle; Cell Transformation, Neoplastic; Cyclins; Flow Cytometry; G2 Phase; Methylnitronitrosoguanidine; Mice; Mice, Inbred BALB C; Microfilament Proteins; Mitosis; Muscle Proteins; Proliferating Cell Nuclear Antigen; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Suppressor Protein p53

An association between low selenium intake and the incidence or prevalence of cancers is well known. Selenium in the form of selenomethionine supplemented in drinking water has been found to be highly effective in reducing tumour incidence and preneoplastic foci during the development of hepatocarcinogenesis in rats in our previous studies. Here, an attempt has been made to investigate whether the dose and form of selenium found to be effective during hepatocarcinogenesis is equally effective in N-methylnitronitrosoguanidine-induced colorectal carcinogenesis in terms of antioxidant defence enzyme systems, DNA chain breaks and incidences of aberrant crypt foci. Treatment with selenomethionine either on initiation or on selection/promotion, or during the entire experiment showed that selenomethionine was most effective in regulating the cellular antioxidant defence systems, DNA chain break control and reducing aberrant crypt foci in the colorectal tissues of rats. Our results also confirm that selenium is particularly effective in limiting the action of the carcinogen during the initiation phase of this colorectal carcinogenesis, just as we found with hepatocarcinogenesis in our previous studies.

Topics: Animals; Antioxidants; Carcinogens; Cell Transformation, Neoplastic; Chemoprevention; Colorectal Neoplasms; DNA Damage; Male; Methylnitronitrosoguanidine; Rats; Rats, Sprague-Dawley; Selenomethionine

The modulatory effects of garlic extract on the in vivo clastogenicity of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a carcinogenic nitrosamine, were evaluated by quantification of micronuclei and chromosomal aberrations in metaphase cells from the bone marrow of male Wistar rats. A single intraperitoneal injection of MNNG (40 mg/kg bodyweight) was found to be clastogenic as revealed by the increased frequency of micronucleated polychromatic erythrocytes and chromosomal aberrations. Pretreatment with aqueous garlic extract (250 mg/kg bodyweight) for 5 days significantly reduced the frequencies of MNNG-induced micronuclei and chromosomal aberrations. The results demonstrate that administration of garlic extract protects against the clastogenic effects of MNNG.

Topics: Animals; Cell Transformation, Neoplastic; Chromosome Aberrations; Garlic; Male; Metaphase; Methylnitronitrosoguanidine; Micronucleus Tests; Mutagenesis; Mutagens; Plant Extracts; Random Allocation; Rats; Rats, Wistar; Stomach Neoplasms

To study the role of superoxide anion (O2.-) in promoting proliferation and transformation of rat liver oval cell strain WB-F344.. WB-F344 cells cultured were stimulated directly by O2.- generated by interaction of xanthine with xanthine oxidase (X-XO). The effect of O2.- in promoting proliferation of WB cells was investigated by using MTT colorimetric analysis, 3H-Tdr incorporation liquid scintillation counter and 3H-Tdr incorporation autoradiography. WB cells initiated with N-methyl-N'-nitro-N-nitrosoguanidine(MNNG) were promoted by stimulating continuously with O2.- of low concentration (X: 100 mumol/L, XO: 0.2 mU/ml). The transformation effect was tested by morphologic observation, karyotype analysis and anchorage-independent growth assay.. Proliferation of WB cells was induced obviously by O2.- of low concentration for only one time. WB cells initiated with MNNG were transformed by action with O2.- of low concentration continuously for 15 d and typical morphologic character of transformed cells was observed. In karyotype analysis the cells chromosome number changed and the frequency of structure aberration raised dramatically. Also the transformed cells could form clone on self-solid culture medium.. The biological effect of O2.- was related closely with its dose; The effects of low concentration in promoting proliferation and transformation of liver oval cells indicate its important role in hepatocarcinogenesis and antioxidation was able to provide a new clue in prevention and cure of hepatoma.

Topics: Animals; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Liver; Methylnitronitrosoguanidine; Rats; Rats, Inbred F344; Stem Cells; Superoxides

Butachlor is a widely used herbicide in Asia and South America. Previous investigations have indicated that it is a suspected carcinogen. To understand more about the biological effects of butachlor on cultured cells and the mechanism(s) of its carcinogenicity, we studied the alteration of the growth characteristics that was induced by butachlor in normal mouse liver cells (BNL CL2). This study demonstrates that butachlor decreases the population-doubling time of BNL CL2 cells, suggesting that it stimulates cell proliferation. To support this finding, a thymidine incorporation assay was conducted and a similar result that butachlor stimulates cell proliferation was elucidated. In addition, we show that butachlor increases the saturation density of the BNL CL2 cells. When combined with the tumor initiator N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), butachlor transforms cells efficiently, as demonstrated by loss of contact inhibition. These findings indicate that butachlor alters the growth characteristics of BNL CL2 cells and suggest that butachlor may induce malignant transformation through stimulation of cell proliferation, alteration of cell cycle regulation, and suppression of cell density-dependent inhibition of proliferation.

Topics: Acetanilides; Animals; Carcinogens; Cell Division; Cell Line; Cell Transformation, Neoplastic; Dose-Response Relationship, Drug; Drug Interactions; Hepatocytes; Herbicides; Methylnitronitrosoguanidine; Mice; Models, Animal; Thymidine

Although the alternative splicing of various genes is a common event in human tumors, the mechanisms behind it have not been characterized. We hypothesized that the expression of splicing regulatory factors would be changed during cellular transformation. Gene expression of three splicing regulatory factors, alternative splicing factor/splicing factor 2 (ASF/SF2), heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2) and the 65 kDa subunit of U2 small nuclear ribonucleoprotein particles auxiliary factor (U2AF(65)), were examined by northern blotting in a two-step chemical transformation model. This in vitro model is composed of BALB/3T3 cells and a BALB/3T3-derived N-methyl-N-nitro-N-nitrosoguanidine (MNNG)-initiated cell line (MT-5). MT-5 cells can be transformed on exposure to 12-O-tetradecanoylphorbol-13-acetate (TPA). ASF/SF2 mRNA levels were decreased 2-fold in both MNNG-initiated cells and TPA-induced transformed cells compared with the normal parental cells, whereas hnRNP A2 mRNA expression did not significantly change between these three types of cells. U2AF(65) mRNA levels were markedly increased ( approximately 4.7-fold) associated with progression of cellular transformation. Moreover, RT-PCR analysis showed that distinct forms of ASF/SF2 mRNA were present in the MNNG-initiated cells and TPA-induced transformed cells but not in the parental cells. These findings indicate that ASF/SF2 or U2AF(65) gene expression is altered during in vitro two-step chemical transformation. The data suggest that the differential expression of splicing regulatory factors is one cause of aberrant expression of alternatively spliced mRNAs encoded by various genes in tumor cells.

Topics: 3T3 Cells; Animals; Base Sequence; Cell Line; Cell Transformation, Neoplastic; DNA Primers; Methylnitronitrosoguanidine; Mice; Mice, Inbred BALB C; Nuclear Proteins; RNA Splicing; RNA-Binding Proteins; Serine-Arginine Splicing Factors; Tetradecanoylphorbol Acetate

The MNNG-exposed Bloom syndrome (BS) B-lymphoblastoid cell population (BS-MNNG), when analyzed for aberrant genetic variations, showed an illegitimate rearrangement at the TCR-gamma gene and hypermethylation at the c-myb protooncogene. The TCR-gamma rearrangement involved a Vgamma9 segment corresponding to a 4 kb band detected with a Jgamma-specific probe in HindIII-digested DNA samples from BS-MNNG cells only. These variations were not shown by unexposed BS cells or both MNNG-exposed and unexposed normal (GA3) B-lymphoblastoid cells.

Topics: B-Lymphocytes; Bloom Syndrome; Carcinogens; Cell Line, Transformed; Cell Transformation, Neoplastic; DNA Methylation; Gene Rearrangement, gamma-Chain T-Cell Antigen Receptor; Humans; Methylnitronitrosoguanidine; Oncogenes

MAPK (Mitogen-Activated Protein Kinase) is one of the elements of kinase cascades (MAPK, MEK-MAP kinase, kinase, Raf-1, Ras) regulating cellular proliferation and differentiation processes. It seems that the changes in its number and activity may be the factor having influence on carcinogenesis. In some human carcinomas a significant increase of its activity is observed, in others a decrease of its activity is described. Our research aimed at the evaluation of the dynamics of precancerous and cancerous changes in the stomach stump in rats after the experimental, partial stomach resection. Apart from histological and ultrastructural examination we also determined the activity of the sub-unit p42 MAP kinase. The material comprised segments of gastric mucosa of the stomach stump of 15 rats after subtotal gastrectomy. Part of the rats after the procedure were administered carcinogen orally (MNNG). On the histological and ultrastructural examination we used routine methods, the activity of MAP kinase was determined by western-blotting method with the use of IgG against MAPK p42, Santa Cruz #154). In 8 examined rats we observed the increase of MAP kinase activity. We established probable correlation (without statistical analysis, regarding miserly material) between the increase of MAPK activity and histological and ultrastructural changes. Among three cases diagnosed as adenoma tubulare in two we observed the increase of MAPK activity. A clear increase of this kinase was also present in the stomach stump of a rat, which was diagnosed as adenocarcinoma. On the basis of our research carried so far we think that the increase of the MAPK activity may be one of the causes of the neoplasm development. It seems important to obtain the confirmation of our results and to establish a possible usefulness of MAPK activity determination as a prognostic indicator in case of the neoplasm of stomach stump.

Topics: Animals; Calcium-Calmodulin-Dependent Protein Kinases; Carcinogens; Cell Transformation, Neoplastic; Gastrectomy; Gastric Mucosa; Gastric Stump; Humans; Male; Methylnitronitrosoguanidine; Rats; Rats, Wistar; Risk Factors; Stomach Neoplasms

Deoxyribonucleoside triphosphate (dNTP) pools were measured in normal BALB/c3T3 cells, transformation-treated cells and transformed cells with reverse-phase HPLC. The fluctuation of dNTP pools was similar after cells were treated with alkylating mutagens glycidyl methacrylate (GMA) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The gap between (dGTP + dATP) pools and (dTTP + dCTP) pools was greatly intensified. The measurements also indicated that the dNTP pools in transformed cells were quite different from those in normal cells. The results suggest that dNTP pools may play an important role in cell transformation.

Topics: 3T3 Cells; Animals; Bisphenol A-Glycidyl Methacrylate; Cell Transformation, Neoplastic; Chromatography, High Pressure Liquid; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Methylnitronitrosoguanidine; Mice; Mice, Inbred BALB C; Mutagens

To investigate the relationship between p53 gene mutations and mouse skin tumors induced by three-step carcinogenesis.. The exons 5-8 of p53 gene were examined in 37 DMBA-TPA-MNNG induced mouse skin tumors [including 6 mice with papillomas, 15 mice having well differentiated squamous cell carcinomas (SCCI) and 16 mice with intermediately differentiated squamous cell carcinomas (SCC II)].. No p53 gene mutation was detected in the papilloma group, whereas 25.8% (8/31) of the SCC group had p53 gene mutations (4/15 of the SCC I mice and 4/16 of the SCC II mice). A total of 9 mutations were found in 8 mice with SCC, of which 7 were located in exon 8 and 7 were G-->A transitions.. The p53 gene mutations occurred during the process of malignant transformation from papilloma to SCC induced by three step carcinogenesis.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; Exons; Genes, p53; Methylnitronitrosoguanidine; Mice; Mice, Inbred SENCAR; Papilloma; Point Mutation; Skin Neoplasms; Tetradecanoylphorbol Acetate

The ability of neighbouring normal cells to inhibit proliferation of transformed cells is regarded as the classical mode of intercellular control of potential tumour cells. This mechanism, however, only controls the pool size of transformed cells, but does not impair their survival. We have recently shown that cells transformed by biological agents are subject to a novel control system: transforming growth factor beta (TGF-beta) induces normal cells to release factors that mediate apoptosis specifically in transformed cells. Here we show that cells transformed by chemical carcinogens are also subject to this dominant control mechanism. The number of foci induced by methylcholanthrene, N-methyl-N'-nitro-N-nitrosoguanidine or quercetin was significantly reduced when the cultures were treated with TGF-beta. Established lines of chemically transformed cells proved to be sensitive to induction of apoptosis by neighbouring normal cells in the presence of TGF-beta. This finding demonstrates that sensitivity to induction of apoptosis is a general feature of transformed cells, irrespective of the transforming agent. It is particularly relevant for chemical carcinogenesis. As transformed cells were shown to trigger induction of their own apoptosis, the acquisition of resistance to this process may be a central regulatory step in carcinogenesis in vitro and possibly also in vivo. This study may help to elucidate mechanisms that protect transformed cells at an early stage of tumour progression that has until now not been the focus of investigation.

Topics: Animals; Apoptosis; Carcinogens; Cell Communication; Cell Count; Cell Division; Cell Line, Transformed; Cell Transformation, Neoplastic; DNA Fragmentation; Fibroblasts; Methylcholanthrene; Methylnitronitrosoguanidine; Quercetin; Transforming Growth Factor beta; Tumor Stem Cell Assay

The mutation frequency of pS189 shuttle vector plasmids is higher in human oral keratinocytes (NHOK) immortalized with cloned human papillomavirus-16 (HPV-16) genome than in primary normal NHOK (NHOK). To determine whether oncoproteins E6 and E7 of HPV-16 are responsible for the higher mutation frequency of the plasmids, we measured the mutation frequency in NHOK and in NHOK expressing the HPV-16 oncogenes (E6, E7, or E6 plus E7). We also measured the mutation frequency in NHOK expressing the E6 or E7 proteins of the non-oncogenic HPV-6b. The mutation frequency, either background or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced, in NHOK expressing the HPV-16 oncoproteins (E6, E7, or E6 plus E7) was significantly higher than in NHOK. The HPV-16 oncogenes did not alter the nature of the MNNG-induced mutations (G:C-->A:T), but increased the frequency of deletions and insertions with or without MNNG. The background or MNNG-induced mutation frequency in NHOK expressing the HPV-6b E6 or E7 proteins was the same as in NHOK. NHOK and NHOK expressing HPV6b-E6 or E7 were able to arrest the cell cycle and enhance cellular p53, p21(WAF1/CIP1), and Gadd45 levels when exposed to MNNG, whereas NHOK expressing the HPV-16 E6 oncogene did not demonstrate. NHOK expressing HPV-16 E7 were able to enhance cellular p53, p21(WAF1/CIP1), and Gadd45 levels, but failed to arrest cell cycle progression when exposed to MNNG. These data indicate that HPV-16 E6 and E7 oncogenes are mutagenic in human oral keratinocytes and enhance the mutagenic effect of MNNG. However, the E6 and E7 proteins of the 'low risk' HPV-6b did not demonstrate such an ability.

Topics: Cell Cycle; Cell Transformation, Neoplastic; Cells, Cultured; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; GADD45 Proteins; Humans; Intracellular Signaling Peptides and Proteins; Keratinocytes; Methylnitronitrosoguanidine; Mouth; Mutagens; Oncogene Proteins, Viral; Papillomavirus E7 Proteins; Plasmids; Proteins; Repressor Proteins; Tumor Suppressor Protein p53

In the BALB/c-3T3-cell transformation system, the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) exposure on the appearance of transformed cells was examined in order to investigate the mechanisms of in vitro tumor promotion. Optimal duration of TPA exposure on N-methyl-N'-nitro-N-nitrosoguanidine(MNNG)-initiated cells was at least 11 days. To investigate the effect of transformation frequencies of altering inoculating cell density at the replating of MNNG-exposed cells and of altering the time of starting TPA exposure, MNNG-exposed cells were replated at various inoculum sizes. With lower inoculum sizes (1 x 10(3) to 3 x 10(4) cells/dish), maximum TPA-induced transformation occurred for TPA commencement at confluence, while with higher inoculum size (1 x 10(5) cells/dish), maximum transformation frequency was observed when TPA exposure was started on day 7 after replating, being some 2 days after confluence. This may suggest that there are different mechanisms involved, depending on inoculum size, and that these may involve cell-cell interactions (at lower inoculum) and mutation expression periods (at higher inoculum). By means of redispersion experiments, it was demonstrated that the appearance of transformed cells begins on about day 7 after replating at a cell density of 1 x 10(4) cells/dish. These results suggest the usefulness of the replating method for optimizing transformation in the BALB/c-3T3-cell transformation assay, and provide insight into the time frame of expression of MNNG-initiated transformants and TPA-induced expansion of these transformants.

Topics: 3T3 Cells; Animals; Carcinogens; Cell Count; Cell Line, Transformed; Cell Transformation, Neoplastic; Methylnitronitrosoguanidine; Mice; Mice, Inbred BALB C; Tetradecanoylphorbol Acetate; Time Factors

Tissue inhibitor of metalloproteinases-1 (TIMP-1), a natural inhibitor of matrix metalloproteinases (MMPs), is known to inhibit invasion and metastasis of tumor cells. In the present study we examined anti-tumor promoter activity of TIMP-1 and its effect on in vitro cell transformation using BALB/3T3 cells in low serum culture medium. In the dye transfer assay the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) continuously blocked gap-junctional intercellular communication (GJIC) of BALB/3T3 cells in confluent phase. TIMP-1 did not prevent transient inhibition of GJIC induced by TPA, but it quickly restored the reduced GJIC level to the control level. The recovery of GJIC was dependent on the concentration of TIMP-1 from 1 to 1000 ng/ml. In an in vitro two-stage transformation assay in which BALB/3T3 cells were treated with 0.5 microg/ml N-metyl-N'-nitro-N-nitrosoguanidine as initiator and 100 ng/ml TPA as promoter, TIMP-1 at concentrations > 10 ng/ml inhibited the focus formation of transformed cells by approximately 60%. TIMP-2 and a synthetic MMP inhibitor showed a similar inhibitory activity on in vitro cell transformation. Furthermore, zymographyic analysis showed that TPA treatment of BALB/3T3 cells induced secretion of gelatinase B and stromelysin-1 into the culture medium. These results indicate that TIMP-1 and TIMP-2 have inhibitory activity on in vitro transformation of cells. It seems likely that TPA-inducible MMPs are involved in carcinogenesis and TIMPs have a protective role against carcinogenesis in vivo.

Topics: 3T3 Cells; Animals; Antineoplastic Agents; Cell Communication; Cell Transformation, Neoplastic; Fibroblasts; Gap Junctions; Metalloendopeptidases; Methylnitronitrosoguanidine; Mice; Protease Inhibitors; Tetradecanoylphorbol Acetate; Tissue Inhibitor of Metalloproteinase-1; Tissue Inhibitor of Metalloproteinase-2

Two of the most representative halogenated aliphatic hydrocarbons, 1,2-dibromoethane and 1,1,2,2-tetrachloroethane, were tested in the two-stage cell transformation model for analysing the promoting ability. Both of these compounds had previously been found to exert genotoxic effects, probably acting as moderate initiators. BALB/c 3T3 cells were initiated with subtransforming doses of N-methyl-N-nitro-N-nitrosoguanidine or 3-methylcholanthrene and then exposed to a chronic treatment with different non-transforming dosages of the two haloalkanes. 1,1,2,2-Tetrachloroethane did not exert any promoting activity in that system. By contrast, significant promoting effects by 1,2-dibromoethane were observed both in cells treated with N-methyl-N-nitro-N-nitrosoguanidine and in cells treated with 3-methylcholanthrene. Promotion of the transformation process initiated with 3-methylcholanthrene was detectable when confluent cells in the chemical-treated plates were replated in the level-II amplification test. This experimental procedure allowed cells to perform further rounds of replications and transformed foci to became detectable. Results gave evidence for a promoting role of 1,2-dibromoethane in multistep carcinogenesis, probably responsible for the higher oncogenic ability of this compound with respect to 1,1,2,2-tetrachloroethane.

Topics: 3T3 Cells; Animals; Carcinogens; Cell Transformation, Neoplastic; Dose-Response Relationship, Drug; Ethane; Ethylene Dibromide; Hydrocarbons, Chlorinated; Methylnitronitrosoguanidine; Mice; Tetradecanoylphorbol Acetate

HPV-immortalized human oral keratinocytes can convert to tumorigenic cells when exposed to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), but normal human oral keratinocytes cannot transform with a similar exposure. The different responses of these cells could be due to different genetic stability of cells. In as much as genetic stability is determined by cell cycle control and of repair of damaged DNA, we studied the effect of MNNG exposure upon cell cycle progression, expression of p53, WAF1/CIP1 and gadd45, and the mutation frequency of a shuttle vector pS189 in normal human oral keratinocytes, in HPV-immortalized oral keratinocytes, and in an oral cancer cell line expressing mutant p53. Normal cells demonstrated transient cell cycle arrest after exposure to MNNG, but the other tested cells did not. While MNNG exposure significantly increased the levels of intranuclear wt p53 protein and the expression of WAF1/CIP1 and gadd45 genes in normal cells, it did not alter them in the immortalized and cancer cells. The mutation frequency of pS189 plasmid was significantly lower in normal cells than in the other tested cells. These data indicate that malignant conversion of HPV-immortalized oral keratinocytes may, in part, be associated with the cells' genetic instability. The genetic instability may be due to cells' (1) inability to accumulate intranuclear wt p53 to a threshold level at which p53 upregulates the transcription of WAF1/CIP1 and gadd45, resulting in the loss of cell cycle control and (2) inefficient repair of DNA damage caused by genotoxic agents.

Topics: Carcinogens; Cell Cycle; Cell Line, Transformed; Cell Nucleus; Cell Transformation, Neoplastic; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; DNA Damage; GADD45 Proteins; Genetic Vectors; Humans; Intracellular Signaling Peptides and Proteins; Keratinocytes; Methylnitronitrosoguanidine; Mouth; Papillomaviridae; Point Mutation; Proteins; RNA, Messenger; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Up-Regulation

The mechanisms of the tumor promoting activity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were studied using as in vitro model the enhancement ('promotion') of malignant transformation of C3H/M2 mouse fibroblasts induced by N-methyl-N'-nitro-N-nitrosoguanidine or 3-methylcholanthrene. In this assay, the promoting effect of TCDD was maximal at a very low concentration of 1.5 pM and was comparable to the effect of the reference tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA, 0.25 microg/ml). The role of reactive oxygen species in the promoting action was investigated: mannitol, a scavenger of hydroxyl radicals, or antioxidants, i.e. ascorbic acid plus alpha-tocopherol, abolished the in vitro promoting effects of TPA and TCDD. Furthermore, the involvement of protein kinase C (PKC) activation was studied: the protein kinase inhibitor H-7 markedly reduced the in vitro promoting activity of TPA but did not affect the promotion by TCDD. In accord with these results, TPA, but not TCDD, enhanced the PKC activity in C3H/M2 fibroblasts. Since the TPA-mediated activation of PKC was not affected by ascorbate plus alpha-tocopherol, it is concluded that the antioxidants interfere with tumor promotion at a step beyond PKC activation. Thus, the results suggest that the enhancement of malignant cell transformation by TPA and TCDD is dependent on a common mechanism, possibly induced by oxygen radicals, and, in addition, on further mechanisms that may involve agent-specific signalling pathways (e.g. PKC activation by TPA).

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Anticarcinogenic Agents; Antioxidants; Ascorbic Acid; Carcinogens; Cell Transformation, Neoplastic; Drug Interactions; Enzyme Inhibitors; Fibroblasts; Isoquinolines; Mannitol; Methylnitronitrosoguanidine; Mice; Mice, Inbred C3H; Piperazines; Polychlorinated Dibenzodioxins; Protein Kinase C; Reactive Oxygen Species; Tetradecanoylphorbol Acetate; Vitamin E

Epidemiologic study has shown the association of nitrosamide compounds with the high incidence of stomach cancer in south China. To study the mechanism of gastric carcinogenesis, we have established an immortalized human gastric epithelial cell line GES-1. GES-1 cells and the normal gastric tissues were treated with different concentrations of MNNG for 24 hours. Point mutation at codon 12 of c-Ha-ras gene was found in cells and tissues (43%) as demonstrated by PCR-RFLP. Rearrangement of c-met gene and amplification of c-erbB2 gene were detected by Southern blot assay on the MNNG treated GES-1 cells. The results indicate that MNNG treatment was intimately associated with the activation of certain oncogenes. H-ras and c-met genes, serving as early targets of carcinogens may play important role in the carcinogenesis of human gastric epithelial cells.

Topics: Cell Transformation, Neoplastic; Cells, Cultured; Epithelial Cells; Gastric Mucosa; Gene Amplification; Gene Expression Regulation, Neoplastic; Gene Rearrangement; Genes, erbB-2; Genes, ras; Humans; Methylnitronitrosoguanidine; Point Mutation; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; Proto-Oncogenes

An electron microscopic, immunocytochemical, and enzyme cytochemical analysis of the previously established oval cell lines OC/CDE 6 and OC/CDE 22 was performed to characterize the phenotype and differentiation patterns of long-term cultures of oval cells. It was found that alpha-fetoprotein, albumin, and cytokeratin 19 are present in all cultured cells. This indicates that oval cells constitute a population of immature cells expressing features of the antigenic phenotype of both the hepatocyte and bile ductular cell lineages. An electron microscopic examination revealed a gradual alteration in the ultrastructure of oval cells toward hepatocyte-like cells. The majority of the oval cells were positive for glucose-6-phosphatase activity. A particularly striking observation was that oval cells were heterogeneous in terms of peroxisome content. Only about 50% of the oval cells had peroxisomes in the cytoplasm, these cells probably being part of the hepatocyte lineage. The other cultured cells did not reveal catalase activity and probably represented cells committed to the bile ductular cell lineage. An addition of clofibrate to the culture medium resulted in a marked peroxisome proliferation in all oval cells, indicating that oval cells might be able to change their differentiation pathway depending on environmental influence toward the hepatocyte lineage. It is most intriguing that in oval cells with abundant cytoplasm peroxisome proliferation was accompanied by proliferation of the smooth endoplasmic reticulum (this is a morphological marker of mature hepatocytes). Taken together, our findings suggest that within the oval cell lines OC/CDE 6 and OC/CDE 22 cells undergoing a morphological and functional differentiation along the hepatocyte and bile ductular cell lineages are present.

Topics: 3,3'-Diaminobenzidine; Albumins; alpha-Fetoproteins; Animals; Antigens, Neoplasm; Cell Differentiation; Cell Transformation, Neoplastic; Choline Deficiency; Clofibrate; Ethionine; Glucose-6-Phosphatase; Immunohistochemistry; Keratins; Liver; Liver Neoplasms, Experimental; Methylnitronitrosoguanidine; Microscopy, Electron; Phenotype; Rats; Rats, Sprague-Dawley; Staining and Labeling; Vimentin

In order to improve the in vitro transformation assay using BALB/3T3 cells, which is routinely conducted with minimal essential medium (MEM) containing 10% fetal calf serum (FCS), we examined the effect of a new medium after the cells had been treated with carcinogens. Preliminary experiments suggested that the use of T medium (modified DME/F-12) supplemented with insulin, transferrin, ethanolamine and sodium selenite plus 2% FCS resulted in a high transformation frequency. The present study confirmed that this medium was very efficient at inducing transformation foci. Transformation frequency was highest when 12-O-tetradecanoylphorbol-13-acetate (TPA) was started from 1 week after the carcinogen treatment. According to the new protocol using this medium, the transformation frequency was five times higher and transformation foci appeared much earlier than with the protocol using usual MEM plus 10% FCS medium. In addition, we tested several typical carcinogens and a promoter in order to confirm the applicability of the new protocol to the two-stage transformation assay. N-Methyl-N'-nitro-N-nitro-soguanidine (MNNG) and benzo[a]pyrene, but not noncarcinogenic benzo[e]pyrene, induced transformation foci. In the metabolic activation system dimethylnitrosamine gave a highly positive transformation. Okadaic acid, a non-TPA-type tumor promoter, enhanced transformation of MNNG-initiated cells. These studies demonstrate that the use of the proposed medium drastically improves transformation frequency in the two-stage in vitro transformation assay.

Topics: 3T3 Cells; Animals; Cell Transformation, Neoplastic; Culture Media; Dimethylnitrosamine; Methylnitronitrosoguanidine; Mice; Tetradecanoylphorbol Acetate

The cytotoxic effects and the transforming properties of two fungicides, metalaxyl and zineb, whose mutagenic or carcinogenic activity has not been clarified yet, were analyzed in the in vitro BALB/c 3T3 cell transformation test both in the presence and in the absence of an exogenous metabolizing system. Zineb was completely detoxified when the exogenous metabolizing system was added to the target cells to increase their inherent metabolic capacity. Metalaxyl induced cell transformation at any assayed dosage, i.e., 500, 250, and 50 micrograms/ml, in the presence of bioactivation, and at the highest dosage (500 micrograms/ml) in the absence of bioactivation. The transforming effect was detectable only in the level-II transformation cultures and it was likely linked to the induction of additional cell proliferation which allowed obtaining the transformation amplification in these experimental conditions.

Topics: 3T3 Cells; Alanine; Animals; Benzo(a)pyrene; Biotransformation; Carcinogens; Cell Survival; Cell Transformation, Neoplastic; Fungicides, Industrial; Methylnitronitrosoguanidine; Mice; Mice, Inbred BALB C; Microsomes, Liver; Zineb

The histological changes occurring in the esophageal mucosa of shrews (Suncus murinus) after N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment were investigated sequentially. Six-week-old female shrews were given a 50 micrograms/ml MNNG solution as drinking water for 30 weeks, and 5 selected at random were killed at 10 and 20 weeks of age, and thereafter at 5-week intervals until 45 weeks of age. Controls were killed at 45 weeks of age. The MNNG-induced esophageal lesion in shrews began from basal cell hyperplasia at 20 weeks of age, followed by dysplasia occurring at 25 weeks of age, then progressed toward intraepithelial carcinoma to invasive squamous cell carcinoma at 35 weeks of age. Apparent sequential dysplasia-carcinoma transition was seen. Papillomas were seen from 25 weeks of age but there was no evidence of papilloma-carcinoma sequence. Five MNNG-untreated shrews killed at the end of the experiment were free of esophageal tumors.

Topics: Animals; Carcinoma in Situ; Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; Disease Models, Animal; Esophageal Neoplasms; Female; Immunoenzyme Techniques; Methylnitronitrosoguanidine; Papilloma; Shrews

Adenocarcinoma, adenoma and dysplasia of the stomach of adult Wistar rats were induced following administration of MNNG for 10 days by gavage when they were new-born. The incidence of the three types of pathological lesions at the dose of 0.4 mg MNNG per rat being 39%, 50% and 100% respectively. Induction of gastric adenocarcinoma is dose-dependent. The incidence of adenocarcinoma in male rats being significantly higher then that of female rats (P < 0.02). 23 of 33 (70%) induced cancers were found in the gastric antrum. By the use of DNA cotransfection technique and the assay of carcinogenicity in the Balb/c nude mice, it was also found that the DNA of 4 of the 6 induced gastric carcinomas could transform NIH/3T3 cells into malignant cells, an indication that the induced cancer DNA contains transforming gene.

Topics: 3T3 Cells; Adenocarcinoma; Animals; Animals, Newborn; Cell Transformation, Neoplastic; Disease Models, Animal; DNA, Neoplasm; Female; Male; Methylnitronitrosoguanidine; Mice; Mice, Inbred BALB C; Mice, Nude; Rats; Rats, Wistar; Stomach Neoplasms; Transfection

Rats carrying the Eker tumor susceptibility mutation are genetically predisposed to renal cell carcinoma. Rats heterozygous for the Eker mutation (Eker carriers) develop multiple bilateral renal cell carcinomas by the age of 1 year. Using an in vitro rat kidney epithelial (RKE) transformation assay developed in our laboratory, proximal tubule cells derived from known Eker rat carriers (+/ek) and non-carriers (+/+) were exposed to the carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), to determine if cells derived from Eker carriers were more susceptible to in vitro transformation than cells derived from non-carrier animals. The percent transformation frequency following MNNG treatment was 7.5-fold higher in cells derived from carrier animals when compared to cells from non-carrier animals. This increased susceptibility to transformation due to inheritance of the Eker mutation is consistent with a predisposition resulting from inactivation of a tumor suppressor gene. The increased susceptibility of kidney epithelial cells carrying the Eker mutation may prove useful in the further development of the RKE transformation assay as a sensitive tool to identify potential renal carcinogens. In addition, because transformation frequency in the RKE assay measures a very early step in multistage transformation, these results also suggest that alterations (by Loss of Heterozygosity or mutation) at the Eker tumor susceptibility locus are an early event in the development of renal tumors in the rat.

Topics: Animals; Carcinoma, Renal Cell; Cell Transformation, Neoplastic; Cocarcinogenesis; Epithelial Cells; Epithelium; Female; Kidney Neoplasms; Kidney Tubules; Male; Methylnitronitrosoguanidine; Mutation; Rats; Rats, Mutant Strains

We immortalized oral keratinocytes by transfecting them with recombinant human papillomavirus (HPV) type 18 DNA and established three cell lines. These lines were morphologically different from their normal counterpart, contained integrated entire HPV-18 DNA, and expressed the viral E6/E7 genes. The cells contained less p53 protein and more c-myc mRNA than normal cells. However, they proliferated only in keratinocyte growth medium (KGM) containing low calcium and were not tumorigenic in nude mice. To test the hypothesis that tumors result from the combined effect of a "high-risk" HPV and chemical carcinogens in the human oral cavity, we exposed the immortalized cells to the chemical carcinogen N-methyl-N'-nitro-N-nitrosoguanidine. Three chemically transformed cell colonies were isolated. These cells (a) proliferated well in both KGM and Dulbecco's modified minimum essential medium containing physiological levels of calcium; (b) were capable of proliferating in nude mice; (c) contained intact, integrated HPV-18 sequences; (d) transcribed substantially more HPV-18 E6/E7, transforming growth factor-alpha, and c-myc than the immortalized counterpart; and (e) contained, like the immortalized counterpart, less wild-type p53 protein and DCC message. These data indicate that human oral keratinocytes can be transformed by sequential exposure of normal keratinocytes to a "high-risk" HPV and chemical carcinogens.

Topics: Animals; Base Sequence; Cell Division; Cell Line, Transformed; Cell Transformation, Neoplastic; Cell Transformation, Viral; DNA, Viral; ErbB Receptors; Gene Expression; Genes, myc; Genes, p53; Humans; Keratinocytes; Methylnitronitrosoguanidine; Mice; Mice, Nude; Molecular Sequence Data; Mouth Neoplasms; Papillomaviridae; RNA, Messenger; RNA, Viral; Transcription, Genetic; Transfection; Transforming Growth Factor alpha

Clonal lines of transformed rat liver epithelial cells, derived from a single population of cloned diploid rat liver epithelial (stem-like) cell line (WB-F344) by exposure in vitro to N-methyl-N'-nitro-N- nitrosoguanidine (MNNG), produce hepatocellular carcinomas, hepatoblastomas and adenocarcinomas in syngeneic rats (Tsao and Grisham, Am. J. Pathol., 127, 168-181, 1987). In this study we show that these clonal lines demonstrate near-diploid (GN clones) or near-triploid (GP clones) aneuploidy and the universal occurrence of non-random chromosomal abnormalities. Marker chromosomes that involved four autosomes--a non-reciprocal translocation involving chromosomes 1 and 7 (t1q43;7q34), and addition of DNA of unknown origin to the pericentromeric regions of chromosomes 4 and 10--occurred in all of the cells of all transformed clones and in the cells of tumors that grew from them. New marker chromosomes involving the same regions of chromosomes 4 and 7 were found in several cell lines established from independent tumors. The preservation of marker chromosomes in tumor cells in the face of random loss and gain of other chromosomes suggests that these non-random aberrations were necessary for tumor formation. The presence of marker chromosomes was associated with increased expression of the c-myc gene (located at q34 on chromosome 7), the c-H-ras gene (located at q41-43 on chromosome 1) and the c-K-ras and TGF alpha genes (both located at unknown sites on chromosome 4), which we have previously shown to be highly correlated with tumorigenicity in these same transformed clonal lines (Lee et al., Cancer Res., 51, 5238-5244, 1992).

Topics: Animals; Cell Line, Transformed; Cell Transformation, Neoplastic; Chromosome Aberrations; Chromosome Deletion; DNA; DNA, Neoplasm; Epithelium; Genetic Markers; Karyotyping; Liver; Liver Neoplasms, Experimental; Male; Methylnitronitrosoguanidine; Phenotype; Ploidies; Rats; Rats, Inbred F344; Tumor Cells, Cultured

Clonal lines of immortal Syrian hamster cells were previously isolated that either suppressed (supB+) tumorigenicity in hybrids with a malignant hamster cell line (BP6T) or had lost this suppression ability (supB-). Neither line was tumorigenic or showed anchorage-independent growth in normal growth medium. SupB- cells, but not supB+ cells, grew in agar supplemented with the growth factors EGF, PDGF and insulin (EPI), providing a selective assay for the supB- phenotype. After treatment of supB+ cells with either N-methyl-N'-nitro-N-nitrosoguanidine (10-300 ng/ml) or 5-aza-2'-deoxycytidine (25-250 ng/ml), and an expression period of 4-8 weeks, a dose-dependent increase in altered cells that grew in agar supplemented with EPI was observed. Cell lines derived from colonies in agar showed persistent EPI-stimulated growth in agar, and decreased suppression of growth in agar for hybrids with BP6T cells. Thus, carcinogen-induced loss of the tumor suppressor phenotype has been demonstrated.

Topics: Animals; Antineoplastic Agents; Azacitidine; Cell Adhesion; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cells, Cultured; Cricetinae; Decitabine; Embryo, Mammalian; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Insulin; Mesocricetus; Methylnitronitrosoguanidine; Mice; Mice, Nude; Phenotype; Platelet-Derived Growth Factor

M3E3/C3 is a clonal fetal hamster lung epithelial cell line which is used for studies of epithelial differentiation as well as for in vitro toxicologic tests. In this study growth kinetics, xenobiotic metabolism, chromosomal stability and transformation were investigated at increasing culture passage numbers up to 150. Cells of higher passages grew faster and reached higher cell densities than the cells of lower ones. As an indicator of xenobiotic metabolism we measured the activity of 7-ethoxycoumarin-deethylase (ECD), an enzyme belonging to the mixed function oxidase system. Up to passage number 100 the ECD activity strongly increased, followed by a slight decrease in additional passages. The chromosomal stability was assessed by the induction of micronuclei by benzo[a]pyrene (BaP) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). More micronuclei were always detected in cells of higher passages than of lower passages. The capability of cells to be transformed to anchorage independent growth by chemical carcinogens was examined using a soft agar test. After carcinogen exposure with BaP and MNNG, cells of higher passages showed higher transformation frequencies than cells of lower passages. Many cells at passage 150 exhibited an especially high soft agar growth even without carcinogen treatment and were therefore characterized as spontaneously transformed. These results show that metabolic and genetic characteristics of permanently growing cells differ remarkably depending on the culture passage. This has always to be considered when permanently growing cells are used for toxicological studies.

Topics: 7-Alkoxycoumarin O-Dealkylase; Analysis of Variance; Animals; Benzo(a)pyrene; Cell Count; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cellular Senescence; Cricetinae; Cytological Techniques; Epithelial Cells; Feasibility Studies; Fetus; Mesocricetus; Methylnitronitrosoguanidine; Micronucleus Tests; Regression Analysis; Reproducibility of Results

The growth and reproduction complex (grc-) strains of rats have a 70-kilobase deletion in the major histocompatibility complex (MHC)-linked grc-G/C region that is associated with embryonic death, developmental defects, and an increased susceptibility to chemical carcinogens. To study further the effects associated with the deletion, fibroblastic cell lines from grc-, grc+, and grc+/- rat embryos were developed: BIL-derived cell lines are congenic for the MHC and grc, whereas R16-derived cell lines are congenic for the grc alone. In early passages, all cell lines expressed the MHC class I antigen RT1.A, had a diploid chromosome number, and did not display anchorage-independent growth or in vivo tumorigenicity. The grc- cells [median population doubling time (PDT), 47 h] grew more slowly than the grc+ (PDT, 30.5 h) and grc+/- (PDT, 33 h) cells. All cells underwent crisis, but the crisis stage began earlier and lasted longer in the grc- cells. The established grc- cell lines (PDT, 32.5 h) grew faster than the grc+ (PDT, 48.5 h) and grc+/- (PDT, 54 h) cell lines. Two of the three BIL-derived grc- lines that survived crisis became anchorage independent in tissue culture and tumorigenic in histocompatible F1 rats (highly malignant fibrosarcomas) at passages 33 and 48, respectively; by contrast, none of the R16-derived grc- cell lines transformed. None of 8 grc+ or 8 grc+/- cell lines that survived crisis displayed anchorage-independent growth or tumorigenicity under the same conditions up to passage 50. All of the established cell lines, including the two tumorigenic ones, expressed MHC class I antigens. Southern and Northern blot analyses of BIL-derived cell lines before and after crisis showed that they all constitutively expressed H-ras and Rb and that no cell line showed rearrangement, amplification, or overexpression of c-myc, H-ras, Rb, and p53 either before or after crisis. These observations indicate that: (a) the homozygous grc- deletion is necessary but not sufficient for in vitro transformation; (b) another genetic factor(s) required for transformation is linked to, or possibly in, the MHC; and (c) passage through crisis, spontaneous transformation, or carcinogen treatment does not alter the cellular expression of MHC class I antigens or of several oncogenes and tumor suppressor genes.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinogenicity Tests; Cell Adhesion; Cell Division; Cell Line, Transformed; Cell Survival; Cell Transformation, Neoplastic; Female; Fibroblasts; Gene Deletion; Histocompatibility Antigens Class I; Male; Methylnitronitrosoguanidine; Polymorphism, Restriction Fragment Length; Rats

A transformation system utilizing SHE cells was used to study the carcinogenic effects of Chinese wollastonite, a new mineral fiber. Morphological transformation was observed in SHE target cells treated with either wollastonite or MNNG, a known chemical carcinogen. A single treatment of wollastonite (20 ug/ml) induced typical transformation. The transformation rate of SHE cells induced by MNNG followed by several treatments with wollastonite was higher than that induced by the same dose of MNNG only. Electron microscopic observation showed that the ultrastructure of wollastonite-transformed SHE cells was significantly different than that of normal SHE cells, but similar to that of SHE cells transformed by MNNG. The above results suggest that wollastonite may not be simply an environmental carcinogen, but also a possible promoter.

Topics: Animals; Calcium Compounds; Cell Transformation, Neoplastic; Cells, Cultured; Cricetinae; Embryo, Mammalian; Mesocricetus; Methylnitronitrosoguanidine; Silicates

To establish a standardized model for the transformation of rabbit airway epithelial cells, we attempted to transform rabbit tracheal epithelial (RbTE) cells in culture with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). RbTE cells, harvested by enzymatic digestion from male New Zealand white rabbits, were plated onto feeder layers of irradiated 3T3 cells. Control cells proliferated exponentially during the 2nd week of culture and reached the plateau phase by the 3rd week. Cells exposed to MNNG (0.1 microgram/ml) proliferated in a fashion similar to the control cells, except that there was some delay before proliferation began. The clonogenic activity of RbTE cells rapidly decreased in parallel with the increase in cell population equally in the control and MNNG groups. During the late plateau phase, cells exposed to MNNG regained clonogenic activity, and this compartment size expanded with time, whereas the clonogenic activity in control cultures remained below the detectable level. In RbTE cell cultures exposed three times to 0.1 microgram/ml MNNG, large, persistent and proliferating colonies emerged at a frequency of 1-3 x 10(-2) among the surviving clones, whereas all the control cultures eventually became senescent. The MNNG-induced alteration in the growth potential of RbTE cells, i.e., the extended lifespan, and the maintenance and even expansion of clonogenic activity, was similar to that of transformed rat tracheal epithelial cells. However, no immortal cell line could be established from these growth-altered RbTE cells. We therefore concluded that the growth-altered RbTE cells were partially transformed.

Topics: Animals; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Dose-Response Relationship, Drug; Epithelial Cells; In Vitro Techniques; Male; Methylnitronitrosoguanidine; Rabbits; Trachea

Male Wistar rats were treated concurrently with a combination of the carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG; CAS 70-25-7) and the polyamine-synthesis inhibitor alpha-difluoromethylornithine (DFMO) at two different doses of 0.5% and 1.0% (w/v). Experimental groups were treated with (I) MNNG alone (n = 25), (II) MNNG plus 0.5% (w/v) DFMO (n = 25), (III) MNNG plus 1.0% (w/v) DFMO (n = 25), (IV) 1.0% (w/v) DFMO alone (n = 25). Group V represented untreated controls (n = 20). Both the carcinogen and DFMO were administered in drinking water. The treatment time with the carcinogen and DFMO was 35 weeks. After treatment was completed animals were followed for an additional 50 weeks to cover a total observation time of 85 weeks. Significantly fewer animals developed gastric adenocarcinoma in the two groups of animals that received a combined treatment of MNNG plus DFMO compared to animals treated with the carcinogen alone (P < 0.05 and 0.005). No benign or malignant neoplastic lesions were observed in the stomach or duodenum of animals treated with DFMO alone or in untreated controls. It is concluded that concurrent treatment with DFMO prevents the development of malignant gastric epithelial tumors induced by MNNG in rats.

Topics: Adenocarcinoma; Adenoma; Animals; Cell Transformation, Neoplastic; Duodenal Neoplasms; Eflornithine; Male; Methylnitronitrosoguanidine; Rats; Rats, Wistar; Sarcoma, Experimental; Stomach Neoplasms

To detect the carcinogenicity of sterigmatocystin for human stomach, the human fetal gastric cells, cultured in vitro, were treated with extract solution of Aspergillus versicolor culture (0.117mg sterigmatocystin per Kg culture of Aspergillus versicolor). The cells showed random arrangement, loss of contact inhibition and cell polarity. Some transformed foci were present in different numbers and times according to the dosages of the extract solution. The cells with such changes were analysed with a flow cytometer and the results showed that the cell number in S phase of cell cycle distribution was greatly increased. The above changes were similar to those of the cells treated with MNNG, but no such changes were noted in the control group. These results indicated that sterigmatocystin could induce some characteristics of neoplastic transformed cells and that sterigmatocystin may be a carcinogen for human gastric cancer.

Topics: Aspergillus; Cell Transformation, Neoplastic; Cells, Cultured; Fetus; Humans; Methylnitronitrosoguanidine; Sterigmatocystin; Stomach

We previously immortalized human oral keratinocytes by transfection with recombinant human papillomavirus type 16 (HPV-16) DNA and established two cell lines. These transfected cells were morphologically different from the normal counterpart, contained intact HPV-16 DNA in an integrated form, and expressed numerous viral genes. These cells contained lower levels of wild-type p53 protein and higher levels of c-myc mRNAs compared to normal cells. However, they proliferated only in keratinocyte growth medium containing a low level of calcium and were not tumorigenic in nude mice. A HPV-16-immortalized cell line was exposed to either 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone or N-methyl-N'-nitro-N-nitrosoguanidine. Four chemically transformed cell colonies were isolated. These cells proliferated well in Dulbecco's minimum essential medium containing a physiological level of calcium. They contained, similar to the immortalized counterpart, integrated HPV-16 sequences and lower levels of both wild-type p53 protein and DCC messages compared to normal cells. Among the chemically transformed cells, two colonies obtained from 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone exposure demonstrated an enhanced proliferation capacity in nude mice and transcribed a substantially higher amount of HPV-16 E6/E7, epidermal growth factor receptors, and c-myc genes compared with the immortalized counterpart. These experiments indicate that malignant transformation of oral keratinocytes can be caused by a sequential combined effect of "high risk" HPV and tobacco-related carcinogens.

Topics: Base Sequence; Carcinogens; Cell Division; Cell Line, Transformed; Cell Transformation, Neoplastic; Cells, Cultured; Colonic Neoplasms; DNA Primers; ErbB Receptors; Exons; Genes, myc; Genes, p53; Genes, ras; Humans; Keratinocytes; Male; Methylnitronitrosoguanidine; Molecular Sequence Data; Mouth Neoplasms; Mutagenesis; Nicotiana; Nitrosamines; Oligonucleotides, Antisense; Papillomaviridae; Plants, Toxic; Polymerase Chain Reaction; Transforming Growth Factor alpha

Seventy-five clonal populations of morphologically transformed 10T1/2 cells were established from independent Type II and Type III foci that were of spontaneous origin or were induced by the carcinogenic agents N-methyl-N'-nitro-N-nitrosoguanidine, benzo(a)pyrene diol epoxide-I, and 3-methylcholanthrene. Clonal populations were characterized for expression of selected transformation phenotypes, including growth to elevated saturation density before cessation of proliferation, anchorage independence, ability to reconstruct foci when plated in the presence of wild-type 10T1/2 cells, and tumorigenicity. Forty-one % of the clonal populations expressed only the phenotype of morphological transformation, while 20% expressed all of the transformation phenotypes, including tumorigenicity, in addition to morphological transformation. The remaining clonal populations expressed varying combinations of one or more of the four transformation phenotypes. Clonal populations expressing almost all of the 16 possible combinations of the transformation phenotypes were observed, suggesting that the individual phenotypes segregated independently. Morphological transformation alone was a poor indicator of tumorigenicity, correctly predicting the tumorigenic potential of only 37% of the clonal populations. Among morphologically transformed clonal populations, coexpression of anchorage independence correctly predicted the tumorigenicity of 81% and coexpression of reconstruction of foci on a confluent lawn of wild-type cells correctly predicted the tumorigenicity of 91%. The probability that a morphologically transformed clonal population was tumorigenic correlated with the total number of transformation phenotypes expressed. Expression of the transformation phenotypes differed between tumorigenic and nontumorigenic clonal populations but not between clonal populations established from Type II and Type III foci. Tumorigenicity varied among transformed clonal populations that were induced by the different carcinogenic agents or were of spontaneous origin but did not differ between clonal populations established from Type II and Type III foci.

Topics: Animals; Bayes Theorem; Benzopyrenes; Cell Division; Cell Separation; Cell Transformation, Neoplastic; Clone Cells; DNA, Neoplasm; Methylcholanthrene; Methylnitronitrosoguanidine; Mice; Mice, Inbred C3H; Mice, Nude; Phenotype

Epidemiological studies have indirectly linked compounds of chromium, nickel and arsenic to human carcinogenesis. However, there is no evidence that metal compounds can transform human cells to the tumorigenic phenotype in culture. We show here that exposure to 36 microM NiSO4 for 48-96 h results in transformation of an immortal, nontumorigenic, osteoblast-like cell line, HOS TE85, to the tumorigenic phenotype. Continuous passaging following treatment leads to the formation of a few dense foci. The cells isolated and expanded from the foci are morphologically transformed, and form anchorage-independent colonies of the size and abundance comparable to that formed by Kirsten murine sarcoma virus transformed HOS TE85 cells. The transformed cells from tumors in nude mice, have enhanced levels of plasminogen activators and have lost the ability to form model bone matrix on extended culture in the presence of ascorbic acid and beta-glycerophosphate. A number of cell lines have been established from nude mouse tumors. Cytogenetic analysis reveals 16 marker chromosomes and an aberrant chromosome 16. This is the first report of the transformation of a human cell line to tumorigenic phenotype by a metal carcinogen.

Topics: Animals; Cell Division; Cell Line; Cell Survival; Cell Transformation, Neoplastic; Chromosome Banding; Dose-Response Relationship, Drug; Humans; Karyotyping; Methylnitronitrosoguanidine; Mice; Mice, Nude; Neoplasm Transplantation; Nickel; Osteoblasts; Osteosarcoma; Phenotype; Transplantation, Heterologous; Tumor Cells, Cultured

Human xeroderma pigmentosum (XP) fibroblasts were transformed with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The transformed cells, called ASKMN, were immortalized, grew in agar and were tumorigenic in nude mice. A trp-met oncogene was identified in ASKMN cells, after transfection of high molecular weight DNA on 3T3 mouse cells. The ASKMN cells and the 3T3 transformants expressed the 5-kb mRNA transcribed by the tpr-met oncogene and its p65tpr-met phosphorylated protein. Using the polymerase chain reaction (PCR) technique followed by hybridization with synthetic probes or direct sequencing, we showed that the sequence encompassing the 'rearranged breakpoint' was the same as that previously described in the tpr-met oncogene present in the MNNG-HOS cells. However, G to A transitions found in the tpr or met sequences of the ASKMN oncogene, probably the result of the specific mutagenic activity of MNNG, were absent in the MNNG-HOS gene. Apparently normal chromosomes 1 and 7 were identified in the ASKMN cell metaphases using several cytogenetic techniques.

Topics: Animals; Base Sequence; Cell Line; Cell Transformation, Neoplastic; Child; Chromosome Aberrations; Gene Expression Regulation; Humans; Karyotyping; Methylnitronitrosoguanidine; Mice; Molecular Sequence Data; Proto-Oncogene Mas; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-met; Proto-Oncogenes; RNA, Messenger; Xeroderma Pigmentosum

In contrast to the strong association of human papillomavirus (HPV) type 16 with genital malignancies, HPV 6 has been found essentially in benign genital lesions. In these studies we show that HPV type 6 and 16 DNAs behave differently also in their ability to transform NIH 3T3 cells in cooperation with the carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Although we could show that both HPV-6- and HPV-16-transfected genomes were integrated and expressed in NIH 3T3 cells, only the NIH 3T3 cells which contained the HPV 16 genome became fully transformed after MNNG treatment, as assessed by their ability to form colonies in soft agar and to induce tumors in nude mice. NIH 3T3 cells containing the HPV 6 genome and treated with MNNG did not show this potential. Furthermore, we could detect an increased expression of the E7 gene of HPV 16 in the carcinogen-treated cells containing the HPV 16 genome. These studies indicate that the presence of the HPV 16 genome specifically is also an essential step to in vitro cellular transformation.

Topics: 3T3 Cells; Animals; Blotting, Northern; Blotting, Southern; Cell Transformation, Neoplastic; Cell Transformation, Viral; DNA, Viral; Methylnitronitrosoguanidine; Mice; Nucleic Acid Hybridization; Oncogene Proteins, Viral; Papillomaviridae; Papillomavirus E7 Proteins; Transfection

Initiation and promotion in mouse skin carcinogenesis produce multiple benign tumors, squamous papillomas, but only a few squamous cell carcinomas. The spontaneous conversion from the benign to the malignant phenotype occurs over many months and in stages, but induced malignant conversion can be accomplished more rapidly by exposure of papilloma-bearing mice to mutagens or by transfection of papilloma cell lines with specific oncogenes. The analysis of genetic targets responsible for carcinogen-induced neoplastic progression would be facilitated by the development of in vitro models where the process is rapid, focal, and quantitative. To this end, primary newborn mouse keratinocytes were initiated in vitro by the introduction of the v-rasHa oncogene via a defective retrovirus. Recipient cells produce squamous papillomas and have a high proliferation rate in culture medium with 0.05 mM Ca2+, but fail to grow in medium with 0.5 mM Ca2+ which is permissive for growth of malignant keratinocytes. When v-rasHa-keratinocytes were exposed to mutagens in vitro, proliferative foci emerged after culture in 0.5 mM Ca2+ for 4 weeks. These foci stained intensely red with rhodamine stain, could be easily quantitated, and readily incorporated bromodeoxyuridine. Dose-response studies with several mutagens indicated that the number of foci increased with concentration to the point where excessive cytotoxicity developed. Mutagens varied in potency for producing foci in the following order: cis-diamminedichloroplatinum greater than or equal to benzo(a)pyrene diolexpoxide I greater than N-methyl-N'-nitro-N-nitrosoguanidine greater than or equal to 4-nitroquinoline-N-oxide greater than N-acetoxy-acetyl- aminofluorene. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate was inactive in the assay. A subset of cell lines derived from foci produced malignant tumors in vivo, while others were not tumorigenic. Analysis of DNA from cell lines and tumors revealed that most tumorigenic cell lines maintained the v-rasHa genome, whereas the viral sequences were deleted in nontumorigenic cell lines. Immunohistochemical analysis indicated that proliferative foci and quiescent v-rasHa keratinocytes expressed keratin 8, a marker of v-rasHa expression in cultured keratinocytes. Cells in foci, but not v-rasHa control cells, expressed keratin 13, a marker which is strongly associated with the malignant progression of skin tumors in vivo. This in vitro assay provides a quantitative model to

Topics: Animals; Animals, Newborn; Base Sequence; Carcinogens; Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; Cells, Cultured; Epidermal Cells; Epidermis; Genes, ras; Keratinocytes; Keratins; Methylnitronitrosoguanidine; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Oligodeoxyribonucleotides; Papilloma; Polymerase Chain Reaction; Skin Neoplasms; Tetradecanoylphorbol Acetate; Transfection

Cultured rat liver epithelial cells (RLE) transformed with repeated treatments of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) demonstrate many features of the common biochemical phenotype of multidrug resistance (MDR) seen in vivo in 'resistant hepatocytes'. The cells have increased glutathione-S-transferase placental subunit (GST-Yp), gamma-glutamyltranspeptidase (GGT), glutathione (GSH) and glutathione peroxidase and are resistant to MNNG. Phenotypically identical RLE cells spontaneously transformed by selective culture conditions showed low levels of GGT and GST and were not resistant to MNNG. Both chemical and spontaneous transformants are cross resistant to doxorubicin although resistance is consistently greater in chemical transformants. No direct correlation was found between the degree of resistance to doxorubicin and MDR gene expression in either of the chemically or spontaneously transformed RLE cells. These observations suggest that in chemical carcinogenesis, other mechanisms of drug detoxification are involved and that MDR expression is not a consistent feature.

Topics: Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1; Blotting, Northern; Cell Transformation, Neoplastic; Cells, Cultured; Doxorubicin; Drug Resistance; Epithelial Cells; Epithelium; gamma-Glutamyltransferase; Gene Expression; Glutathione; Glutathione Transferase; Liver; Membrane Glycoproteins; Methylnitronitrosoguanidine; Rats; RNA

While a great deal of evidence has directly implicated the importance of O6-alkylation of guanine in the mutagenicity of alkylating agents, evidence demonstrating the oncogenic potential of this lesion has been largely indirect. We have combined a well-studied in vitro neoplastic transformation system (using C3H/10T1/2 mouse cells) with a proven method of gene transfection for expressing the bacterial O6-alkylguanine-DNA alkyltransferase (AT; EC 2.1.1.63) repair genes ada and ogt to generate subclones which possess augmented repair capability toward specific DNA lesions. The products of these genes specifically and differentially repair O6-methylguanine (O6-MeGua), O4-methylthymine (O4-MeThy), and methylphosphotriesters. We show that the level of expression of either the ada or the ogt AT gene in C3H/10T1/2 cells directly correlates with protection against mutation to ouabain resistance by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Subclones expressing 70 fmol of AT per 10(6) cells exhibited a mutation frequency approximately 1/40th of that of clones expressing 15 fmol of AT per 10(6) cells when treated with MNNG at 0.4 micrograms/ml. Protection against mutagenesis by MNNG at 0.8 micrograms/ml, however, did not exceed 12-fold even in subclones expressing greater than 100 fmol of AT per 10(6) cells. As an MNNG dose of 0.6 micrograms/ml was sufficient to saturate more than 95% of the AT activity in any of the clones, the residual mutation frequency may have been caused by unrepaired O6MeGua lesions. In contrast to mutagenesis, protection against neoplastic transformation in vitro, in cells expressing high levels of AT, was most pronounced in cells treated with the highest dose of MNNG used (1.2 micrograms/ml). Low levels of transformation caused by MNNG at 0.4 and 0.8 micrograms/ml were not consistently inhibited in those clones. These data suggest that O6-MeGua formation is of major but not unique significance in the neoplastic transformation of C3H/10T1/2 cells by MNNG.

Topics: Animals; Cell Cycle; Cell Survival; Cell Transformation, Neoplastic; Cells, Cultured; DNA Damage; DNA Repair; In Vitro Techniques; Methylnitronitrosoguanidine; Methyltransferases; Mice; Mutagenesis; O(6)-Methylguanine-DNA Methyltransferase; Transfection

Pancreatic acinar cells were isolated for culture from a young (Y) and an old (O) Brown-Norway or Fischer 344 rat fed an ad libitum (AL) or calorically restricted (CR) diet. The cells were cultured and cellular growth rates were determined as a function of passage number. An overall increase in cellular growth rate and transformation frequency with age and/or AL diet relative to youth as well as a decrease with CR diet were concordant with reported responses in vivo. Transformation frequency was measured in Brown-Norway cells and followed the same pattern as the growth response: AL/O > AL/Y = CR/Y > CR/O. The cellular model is shown to fit the general multistage requirements of the carcinogenic process as well as general age and diet characteristics of pancreatic cancer. This pancreatic acinar cell age-diet approach may prove to be a valuable tool for determining mechanisms of exocrine pancreatic carcinogenesis as well as other disease states; it may also be of utility in in vitro gerontological nutritional and pharmacological studies since some of the age and diet determinants of biological effects appear to be segregable. Propensity of cells from an old and/or AL diet animal for faster growth and for cellular transformation are programmed into the cells by the time of their excision from the animal (as late as 14 months), indicating a heritable component in the model or a mechanism that is dependent upon elements that control gene expression.

Topics: Aging; Animals; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Energy Intake; Male; Methylnitronitrosoguanidine; Models, Biological; Pancreas; Rats; Rats, Inbred BN; Rats, Inbred F344

Helicobacter pylori (HP) has been shown to possibly be a pathogen of gastric carcinoma. HP has urease activity and produces ammonia in the stomach. In this study, the role of ammonia on gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were investigated in rats. After 24 weeks pretreatment with MNNG (83 mg/l), 0.01% ammonia or tap water as a drinking water was administered for 24 weeks. The ammonia-treated rats showed a significantly higher incidence of gastric cancer (percent of animals with tumors and number of tumors per rat). Ammonia would thus appear to have an important role in HP-related human gastric carcinogenesis.

Topics: Adenocarcinoma; Administration, Oral; Ammonia; Animals; Cell Transformation, Neoplastic; Helicobacter pylori; Male; Methylnitronitrosoguanidine; Rats; Rats, Inbred Strains; Stomach Neoplasms

The mechanisms of reversibility of basal cell hyperplasia in the rat forestomach were investigated. Male F344 rats were given an initial single gastric intubation of N-methyl-N'-nitro-N-nitorosoguanidine and then received 2% butylated hydroxyanisole in the diet from the third week to the 26th week. Rats were killed at weeks 26 and 46 after return to basal diet and their forestomachs were removed. Bromouracil deoxyriboside (BUdR) was administered as a single i.p. injection 1 h before death or by osmotic minipump (120 micrograms/h) continuously for 7 days before death. Additional animals were maintained for 2 or 4 weeks after removal of osmotic minipumps to allow assessment of the fate of proliferating populations. In each case BUdR-labeled cells were demonstrated by immunohistochemistry by immunohistochemistry. At week 26, hyperplastic changes were more pronounced than at week 46. Squamous cells above basal cell hyperplasias were strongly labeled even 4 weeks after cessation of continuous BUdR Three-dimensional reconstruction of persisting basal cell hyperplasias showed almost all basal cells limited to a thin sheet in direct contact with the squamous cell layer, occasional separate islands demonstrating differentiation to squamous cells and formation of epidermal cysts. The results thus showed that the mechanism of reversibility of basal cell hyperplasia involves differentiation of basal cells to squamous cells.

Topics: Animals; Bromodeoxyuridine; Butylated Hydroxyanisole; Carcinoma, Basal Cell; Carcinoma, Squamous Cell; Cell Differentiation; Cell Transformation, Neoplastic; Gastric Mucosa; Hyperplasia; Image Processing, Computer-Assisted; Male; Methylnitronitrosoguanidine; Rats; Rats, Inbred F344; Stomach; Stomach Neoplasms

We investigated the ability of overexpression of the c-myc proto-oncogene to potentiate in vitro transformation by model chemical carcinogens. A mouse c-myc gene was introduced to C3H 10T1/2 and Rat 6 embryo fibroblast cell lines via a retroviral vector containing the gene for neomycin resistance. Our present work extends previous findings by showing that individual vectored C3H 10T1/2 clones have enhanced (two-fold to sevenfold) sensitivity to benzo[a]pyrene (BP) and N-methyl-N-nitro-N'-nitrosoguanidine (MNNG). Rat 6 clones acquiring the c-myc gene display various degrees of altered morphology. They form orderly but densely packed cells, grow to higher saturation density, and yield microcolonies in soft agar. The degree of altered growth properties is directly correlated with the level of c-myc expression. Transient exposure of c-myc-expressing clones to BP and MNNG induced the formation of distinct, large colonies in soft agar, whereas the untreated cells formed microcolonies and the parental Rat 6 cells remained single cells in soft agar. We also demonstrated that the degree of responsiveness to chemical carcinogens of the clones correlates with their ability to form microcolonies in soft agar. These cells overexpressing c-myc may be used as a model system to study the interaction between oncogenes and chemical carcinogens in the process of multistage carcinogenesis.

Topics: Animals; Benzo(a)pyrene; Carcinogens; Cell Line; Cell Transformation, Neoplastic; Clone Cells; Embryo, Mammalian; Fibroblasts; Gene Expression Regulation, Neoplastic; Genes, myc; Genetic Vectors; Methylnitronitrosoguanidine; Mice; Mice, Inbred C3H; Moloney murine leukemia virus; Rats

This project examined the potential role of ozone as a respiratory carcinogen by characterizing its ability to induce or modulate the preneoplastic transformation of rat tracheal epithelial cells. The chemical reactivity of ozone and the types of damage it can cause suggest that it may have a role in environmental carcinogenesis. Previous reports have described an increase in the incidence and number of lung tumors per animal in strain A mice exposed to ozone. However, the role of ozone in the development of the tumors has not been clear. Ozone also has been reported to act alone and synergistically with ionizing radiation to induce changes related to neoplasia in primary hamster embryo cells and in the mouse C3H/10T1/2 cell line in culture. Few other studies have examined the direct cytotoxic or transforming effects of ozone after in vivo or in vitro exposure of cells, and no studies have been reported on the comparative effects of ozone on respiratory cells exposed in vivo or in vitro. The induction of early preneoplastic changes in populations of rat tracheal epithelial cells by carcinogens can be detected and quantified in vitro after exposures in vivo or in vitro of tracheal epithelial cells. This cell culture and transformation system was used to characterize the transforming potency of ozone. Tracheal epithelial cells were isolated from Fischer-344/N rats that had been exposed for six hours per day, five days per week for one, two, or four weeks to 0, 0.12, 0.5, or 1.0 parts per million (ppm)* ozone (sea-level equivalents). Cell populations were examined in culture for increases in the frequency of preneoplastic variants. Rats exposed to ozone did not exhibit an increase in the frequency of preneoplastic tracheal cells, although exposed tracheas did exhibit dose-dependent morphological changes. Rat tracheal epithelial cells were given single, 40-minute in vitro exposures to concentrations of ozone that did not result in any detectable decrease in colony-forming efficiency (approximately 0.7 ppm) and to concentrations that resulted in approximately a 40% decrease (approximately 10 ppm). Exposed cultures were examined for increases in the frequency of preneoplastic variants. The results of these experiments, like those for the in vivo experiments described above, suggest that a single ozone exposure does not induce preneoplastic variants of rat tracheal epithelial cells. In contrast, cultures of rat tracheal cells exposed to 0.7 ppm ozone twice weekl

Topics: Animals; Carcinogens; Cell Transformation, Neoplastic; Cells, Cultured; Drug Interactions; Epithelial Cells; Epithelium; Male; Methylnitronitrosoguanidine; Ozone; Precancerous Conditions; Rats; Rats, Inbred F344; Statistics as Topic; Trachea

The development of carcinoma was examined in male Wistar rats (n = 120) exposed to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in the drinking water (83 micrograms/ml) for 16 weeks. After MNNG administration, rats were investigated by endoscopic observation, visualization of microvascular structure, and estimation of lectin binding sites. Changes of bile reflux to the stomach was observed endoscopically at 24 weeks as well as the development of gastric mucosal erosions. Protruding and expansive ulcerating carcinomas developed at 36 weeks and had a microvascular pattern similar to that of human adenocarcinoma. Estimation of lectin binding site and pattern was useful to evaluate the malignant potential of cell proliferation. We postulate that endoscopic observation is valuable in investigating the development of gastric carcinoma, and microvascular structure and lectin binding pattern may be useful to demonstrate the mechanism of growth of gastric carcinoma.

Topics: Animals; Binding Sites; Cell Transformation, Neoplastic; Disease Models, Animal; Gastric Mucosa; Lectins; Male; Methylnitronitrosoguanidine; Protein Binding; Rats; Rats, Inbred Strains; Stomach Neoplasms

A quantitative in vitro transformation assay has been developed for the first time using primary rat kidney epithelial (RKE) cells. RKE cells were grown in a 50:50 mixture of 3T3 conditioned medium and DF8 medium composed of Ham's F-12/DMEM supplemented with ferrous sulfate, vasopressin, transferrin, sodium selenite and 10% fetal bovine serum. Colony forming efficiency of cells plated in this medium was high, ranging from 2.4 to 16%. Normal RKE cells treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) became transformed to a preneoplastic state of enhanced in vitro growth potential and formed large colonies of morphologically altered cells, whereas RKE cells treated with vehicle alone ceased proliferating and/or sloughed off the dish within 4-6 weeks. Relative survival and percent transformation frequency (Tf) of RKE cells exposed to MNNG were inversely proportional and both were dose dependent. MNNG concentration of 0.5, 1.0 and 2.0 micrograms/ml resulted in transformation frequencies of 0.13, 0.37 and 1.1% respectively (n = 4). Morphologically transformed colonies gave rise to cell lines with indefinite growth capacity and neoplastic potential. One of six transformed RKE cell (TRKE) lines injected into nude mice produced adenocarcinomas. This assay represents the first in vitro model for studying mechanisms of chemical transformation of normal kidney epithelial cells and may also be useful as a screen for identifying potential renal carcinogens.

Topics: Animals; Cell Transformation, Neoplastic; Cells, Cultured; Culture Media; Epithelium; Kidney; Methylnitronitrosoguanidine; Rats; Rats, Inbred F344

Tumorigenicity was correlated with levels of expression of the genes for transforming growth factor alpha (TGF-alpha), epidermal growth factor receptor, c-myc, c-H-ras, and c-K-ras in a series of 16 clonally derived transformed liver epithelial cell lines. The clonal lines, which varied in tumorigenicity from 0 to 97%, were established from a phenotypically heterogeneous population produced by repeated exposure of diploid WB-F344 (WB) cells to N-methyl-N'-nitro-N-nitrosoguanidine. Segregation of gene expression with tumorigenicity among clonal lines was determined by correlating rank orders of gene expression by clones relative to expression by wild-type WB cells. Only the expression of the c-myc gene correlated with tumorigenicity among all transformed clones. TGF-alpha gene expression was not correlated with tumorigenicity among all clones, but it was highly correlated with tumorigenicity among clones that expressed the c-myc gene above the median level for all clones (greater than 5-fold the level of expression by WB cells). Even high levels of expression of the TGF-alpha gene (up to 60-fold the level of expression by WB cells) were not correlated with tumorigenicity among the clones expressing the c-myc gene at levels less than 5-fold the level of expression by WB cells. Clones which simultaneously overexpressed both c-myc and TGF-alpha genes at levels above the median levels for all clones were significantly more tumorigenic than were clones which expressed either or both genes at lower than median levels. These results suggest that overexpressed c-myc and TGF-alpha genes cooperate in their association with tumorigenicity. Most of the highly tumorigenic clones that overexpressed c-myc and TGF-alpha also overexpressed the c-H-ras and/or the c-K-ras genes; clones that overexpressed neither of the c-ras genes nor the genes for c-myc and TGF-alpha were not very tumorigenic, while clones that expressed one or both c-ras genes (but not both c-myc and TGF-alpha) were variably tumorigenic over an intermediate range.

Topics: Animals; Blotting, Northern; Cell Line; Cell Transformation, Neoplastic; Densitometry; DNA Probes; ErbB Receptors; Genes, ras; Liver Neoplasms; Male; Methylnitronitrosoguanidine; Neoplasm Invasiveness; Neoplasm Transplantation; Nucleic Acid Hybridization; Poly A; Proto-Oncogene Proteins c-myc; Rats; Rats, Inbred F344; Regression Analysis; RNA; RNA, Messenger; Tumor Necrosis Factor-alpha

The possibility to use the rat visceral yolk sac as a model for the study of processes of cell transformation was studied. Yolk sac teratocarcinomas could be induced using the method of in vitro culture of yolk sacs in a medium containing a direct carcinogen and a tumor promoter with subsequent transplantation under the renal or testicular capsule of syngeneic rats. Biochemical, electron-microscopic and immunohistochemical methods were used to study the characteristic changes that accompanied cellular transformation. It was shown that even a short-term (3 h) exposure of the yolk sac cells to N-methyl-N'-nitro-N-nitrosoguanidine with or without deoxycholic acid in vitro decreased significantly the rate of yolk sac transport and changed their developmental potential with manifestation of carcinogenic antigens (polyclonal keratins, monoclonal vimentin and smooth muscle actin). This cancerous transformation was promoted following their in vivo transplantation into special anatomic sites.

Topics: Animals; Cell Transformation, Neoplastic; Cells, Cultured; Deoxycholic Acid; Endoderm; Female; Immunohistochemistry; Male; Mesonephroma; Methylnitronitrosoguanidine; Neoplasm Transplantation; Pregnancy; Rats; Rats, Inbred Strains; Teratoma; Testis; Tumor Cells, Cultured; Vimentin; Yolk Sac

We have shown previously that the repair of O6-methylguanine can be induced in murine fibroblasts (C3H 10T1/2 cells) by exposure to X rays. The magnitude of the response is less, however, than is observed in the well-characterized adaptive response of various prokaryotes to methylating agents. To determine whether the induction of O6-alkylguanine-DNA alkyltransferase in C3H 10T1/2 cells is sufficient for protection against the genotoxic effects of the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), cells were challenged with MNNG after alkyltransferase induction by 1.5 Gy X rays and assayed for cytotoxicity, mutagenicity, and neoplastic transformation. Preirradiated cells were significantly more resistant to the mutagenic effects of MNNG as scored by formation of ouabain-resistant colonies. The protective effect was greatest in cells challenged with a low dose (0.2 or 0.4 micrograms/ml) of MNNG. Protection against neoplastic transformation by MNNG was also observed, although the protective effect in this case was significant only in cells treated with a high dose (1.0 micrograms/ml) of MNNG. In cells that were preirradiated, there was no reduction in the cytotoxicity caused by MNNG or the chloroethylating agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). These data indicate that alkyltransferase induction in C3H 10T1/2 cells is sufficient to protect cells against some of the genotoxic effects of the alkylating agent MNNG. The data also suggest that formation of O6-alkylguanine may not be the only means by which alkylating agents can transform C3H 10T1/2 cells.

Topics: Animals; Carmustine; Cell Survival; Cell Transformation, Neoplastic; Cells, Cultured; DNA Damage; Enzyme Induction; Methylnitronitrosoguanidine; Methyltransferases; Mice; Mice, Inbred C3H; Mutation; O(6)-Methylguanine-DNA Methyltransferase; X-Rays

The transforming potency of ozone for rat tracheal epithelial (RTE) cells exposed in vivo or in vitro was determined. RTE cells isolated from rats exposed to ozone (0, 0.14, 0.6, or 1.2 ppm, 6 hr/day, 5 days/week for 1, 2, or 4 weeks) showed no increase in the frequency of preneoplastic transformation compared to cells isolated from unexposed rats, although ozone-induced morphologic changes were observed in exposed tracheas. In contrast, preneoplastic variants of RTE cells were induced by multiple, but not single, exposures of RTE cells to ozone in culture. RTE cells exposed biweekly to ozone (approximately 0.7 ppm for 40 min, nine total exposures) had approximately twofold increases in the frequency of preneoplastic transformation compared to that of concurrent controls exposed to air. Single, 40-min exposures to ozone (approximately 1 or approximately 10 ppm) did not induce preneoplastic variants. However, single, 40-min exposures of RTE cells to approximately 10 ppm ozone did result in approximately 40% decreases in colony-forming efficiency. In addition, single, 40-min exposures of RTE cells to approximately 1 ppm ozone reduced the transforming potency of a subsequent exposure to the direct-acting chemical carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). When multiple ozone exposures followed exposure to MNNG (approximately 0.7 ppm ozone for 40 min, nine biweekly exposures), an additive (or possibly a multiplicative) effect of ozone on MNNG-induced preneoplastic transformation was seen. These results demonstrate that ozone can, under some conditions, induce preneoplastic variants of RTE cells. In addition, depending on the sequence or combinations of exposures, ozone can reduce or, possibly, increase, the transforming potency of the carcinogen MNNG for rat tracheal cells in culture.

Topics: Animals; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Drug Interactions; Male; Methylnitronitrosoguanidine; Ozone; Precancerous Conditions; Rats; Rats, Inbred F344; Trachea; Tracheal Neoplasms

The presence of the viable yellow Avy and lethal yellow Ay genes has been demonstrated to accelerate the process of tumorigenesis in mice bearing genetically, chemically or virally initiated cells. This promoting effect has been reported in the liver, lung, breast, bladder and skin. Although it has been demonstrated that the accelerated process is associated with systemic, pathophysiologic alterations such as an alteration in the lipogenic pathway, which in turn leads to obesity, alteration in glucose metabolism and/or random immunologic alterations, no examination of the in vitro 'tumorigenic' susceptibility of the cells of animals bearing the yellow genes has yet been reported. In the current study, spontaneous and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced transformation was determined in skin-derived fibroblasts from C57BL/6N-Avy/a mice (Hy) and their black a/a litter mates (B1-). The growth rate of untreated fibroblasts was greater for those from the yellow mice than from the black, while susceptibility to MNNG toxicity was equivalent. However, the rate of spontaneous and chemically induced transformation was consistently and significantly higher in the cells obtained from the black a/a mice than in those from yellow, Avy/a litter mates. These findings, with others from the literature, suggest that the Avy gene may suppress transformation and carcinogenic susceptibility in specific cells, such as fibroblasts, while the systemic effects of the gene are promotional in other cells.

Topics: Animals; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Genes; Genetic Predisposition to Disease; Methylnitronitrosoguanidine; Mice; Mice, Inbred C57BL; Mutation; Neoplasms, Experimental

Five clonal cell strains of an early-passage normal rat liver epithelial cell line were transformed spontaneously using the protocol of "selective culture" condition. Twelve cell lines were established from the tumors produced after injecting these transformed cells into 1-day-old syngeneic rats. The phenotypic expressions of these spontaneously transformed tumor cell lines were studied and compared to those of cell lines obtained from tumors produced by rat liver epithelial cells transformed by N-methyl-N'-nitro-N-nitrosoguanidine. Like the chemically induced tumor cells, spontaneously transformed tumor cells exhibited phenotypic heterogeneity in the expression of isoenzymes, proto-oncogenes, growth factors and their receptors, and cellular responses to the effect of growth factors. However, unlike the chemically induced tumor cells, these spontaneously induced tumor cells did not express the "resistant phenotypes" characteristic of chemically induced or promoted tumors. Although all the spontaneously induced tumor cell lines expressed variable amounts of transforming growth factor-alpha mRNA, it was not functionally coordinated with the expression of its receptor, the epidermal growth factor receptor. Thus, spontaneously transformed rat liver epithelial cells demonstrate both similarity and diversity in their phenotypic expression when compared to their chemically induced counterpart. This model of spontaneous transformation of cultured rat liver epithelial cells may be useful for the mechanistic study of non-chemically induced carcinogenesis.

Topics: Animals; Blotting, Northern; Cell Division; Cell Line; Cell Transformation, Neoplastic; Epidermal Growth Factor; Epithelium; Liver; Liver Neoplasms; Methylnitronitrosoguanidine; Nucleic Acid Hybridization; Phenotype; Rats; RNA; RNA, Neoplasm; Transforming Growth Factors

To characterize the potential role of high-l.e.t. radiation in respiratory carcinogenesis, the cytotoxic and transforming potency of 5.5 Me V alpha-particles from electroplated sources of 238Pu were determined using primary cultures of rat tracheal epithelial cells. The alpha-particle response was compared to the effects of 280 kVp X-rays and of the direct-acting carcinogen N-methyl-N'-nitro-N-nitrosoguanidine. Increasing the alpha-particle dose caused an exponential decrease in survival with a D37 of 1.6 Gy. X-rays also caused a dose-dependent decrease in survival (D37 = 3.6 Gy) but the survival curve had a significant shoulder. The RBE for cell killing by alpha-particles versus X-rays varied with dose, and ranged between 4 and 1.5 for alpha doses in the range 0.2-4 Gy. At equally toxic doses (relative survival 0.18-0.2), all three agents induced similar frequencies of preneoplastic transformation. For preneoplastic transformation induced by doses of alpha- and X-radiations giving 80 per cent toxicity, an alpha RBE of 2.4 was derived. The similar RBEs for cell killing and for preneoplastic transformation suggest an association between the type or degree of radiation-induced damage responsible for both cell killing and cell transformation.

Topics: Alpha Particles; Animals; Cell Transformation, Neoplastic; Epithelium; In Vitro Techniques; Male; Methylnitronitrosoguanidine; Rats; Rats, Inbred F344; Trachea; X-Rays

The purpose of this study is to elucidate the participation of Paneth cells in experimentally induced adenocarcinoma of the intestine. The rats were fed with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) dissolved in drinking water ad libitum at a concentration of 100 micrograms/ml for 28 weeks. They were sacrificed 12 weeks after the last MNNG administration. A number of tumor cells containing large eosinophilic granules in their supranuclear cytoplasm (Paneth cells) were observed in about 20% of the experimentally induced adenocarcinoma of the small intestine. The granules were stained positively with Lendrum, periodic acid-Schiff, Masson's trichrome, and Mallory's phosphotungstic acid hematoxylin. Ultrastructurally, the granules were round, osmiophilic, and relatively even in size. We compared the morphologic features of the Paneth cell-containing small intestinal adenocarcinomas (Group I) with those without Paneth cells (Group II). Group I was distinguished from Group II by its better differentiation, larger tumor size and lower incidence of calcification. Although Paneth cells are extremely rare in human gastrointestinal carcinomas, twenty percent of MNNG-induced intestinal carcinomas harbor Paneth cells. The neoplastic Paneth cells in experimental carcinomas may differentiate from uncommitted cells in the deeper portion of the crypt.

Topics: Adenocarcinoma; Animals; Cell Transformation, Neoplastic; Intestinal Neoplasms; Methylnitronitrosoguanidine; Rats; Rats, Inbred Strains

The proceeding of malignantly transformed rodent cells in vitro is often accompanied by regular variation of chromosome numbers and structures. In order to clarify the relationship between them, we obtained five clones C1-1-5-from BHL B4, cell line, and analysed their biological characteristics. Results showed a positive correlation between frequency of chromosome 15 monosomy, it is suggested that suppressive gene of malignant phenotype be located on chromosome 15.

Topics: Animals; Cell Line, Transformed; Cell Transformation, Neoplastic; Chromosome Deletion; Cricetinae; Fibroblasts; Gene Expression Regulation, Neoplastic; Lung; Mesocricetus; Methylnitronitrosoguanidine; Monosomy; Neoplasm Transplantation; Phenotype

tsJT60 is a nonlethal temperature-sensitive (ts) mutant of a Fischer rat cell line (3Y1) classified as a G0 mutant; i.e., the ts defect is not expressed within the cell growth cycle but is expressed only between the G0 and S phase. tsJT60 clones transformed with oncogenes such as adenovirus E1A, polyoma large T, polyoma middle T, v-Ki-ras, and LTR activated c-myc, or with a chemical carcinogen N-methyl-N'-nitro-N-nitrosoguanidine, grew well at 34 degrees C. However, most of these clones grew slowly at 40 degrees C, producing many floating dead cells, and some clones were killed at 40 degrees C. When they were cultured under conditions inadequate for growth of untransformed cells, such as high cell density or serum restriction, they were killed at 40 degrees C. These and previous results from SV40- and adenovirus-transformed tsJT60 clones favour the idea that transformed tsJT60 cells occasionally enter the G0 phase and are metabolically imbalanced at 40 degrees C during self-stimulation from the G0 to S phase. We propose that a drug which exclusively block, G0-G1 transition would be cytocidal to transformed cells but cytostatic to normal cells.

Topics: Adenovirus Early Proteins; Animals; Antigens, Polyomavirus Transforming; Antineoplastic Agents; Carcinogens; Cell Division; Cell Line; Cell Line, Transformed; Cell Transformation, Neoplastic; Cell Transformation, Viral; Drug Design; Genes, Lethal; Genes, myc; Genes, ras; Methylnitronitrosoguanidine; Mutation; Oncogene Proteins, Viral; Oncogenes; Oncogenic Viruses; Rats; Rats, Inbred F344; Recombinant Fusion Proteins; Resting Phase, Cell Cycle; Temperature

An in vitro transformation system was established using primary rat tracheal epithelial (RTE) cells cultured in medium with or without feeder layer cells. About 24-48 hours after plating, RTE cells were exposed to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG, 0-0.6 microgram/ml), benzo (a) pyrene (BaP, 0-20 micrograms/ml), or 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK,0-2.0mg/ml). One or two weeks after exposure, enhanced growth variants (EGV) among the RTE cells were selected in serum containing medium by removing either the feeder layer or bovine pituitary extract (BPE) and epidermal growth factor (EGF). The results showed that EGV transformation was induced by MNNG, BaP and NNK, and a dose dependent EGV transformation was observed in the MNNG and NNK experiments. Our experimental results suggest that for the selection of EGV's from among RTE cells exposed to chemical carcinogens, the use of BPE- and EGF-free medium is more sensitive and convenient than is removing the feeder layers.

Topics: Animals; Benzo(a)pyrene; Carcinogens; Cell Transformation, Neoplastic; Epithelial Cells; Methylnitronitrosoguanidine; Nitrosamines; Rats; Trachea

Stromal cells isolated from normal human endometrium were cotransfected in primary culture with pSVc-myc, a plasmid containing a truncated c-myc gene regulated by simian virus 40 promoter, and pRSV neo, a plasmid containing a neomycin resistance gene regulated by Rous sarcoma virus (RSV) promoter. These cells demonstrated properties of transformed cells in vitro, including altered morphology, focus formation, anchorage-independent growth, chromosomal alterations, and tumor formation in athymic mice. When these cells were treated subsequently with a direct-acting carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine, they demonstrated higher colony-forming efficiency in soft agar and reduced tumor latency. Cells transfected with pRSV neo alone exhibited some properties associated with neoplastic transformation, including altered morphology and formation of colonies in soft agar. It is presumed that in normal cells transfected with pRSV neo, RSV long terminal repeats activated cellular genes that normally regulate growth of human endometrial stromal cells.

Topics: Avian Sarcoma Viruses; Cell Adhesion; Cell Line, Transformed; Cell Transformation, Neoplastic; Drug Resistance; Endometrium; Female; Humans; Methylnitronitrosoguanidine; Neomycin; Plasmids; Proto-Oncogene Proteins c-myc; Transfection; Transformation, Genetic; Uterine Neoplasms

Because danthron, though carcinogenic, does not seem to be genotoxic, it and 8 other hydroxyanthraquinones were comparatively investigated for activities associated with tumor promotion, such as stimulation of cell proliferation and enhancement of malignant transformation. The in vivo treatment of primary rat hepatocytes with danthron, aloe-emodin, chrysophanol, and rhein resulted in a 2-3-fold increase of DNA synthesis, lucidin and purpurin were less active, and emodin, purpuroxanthin, and alizarin were essentially inactive. In addition, danthron, rhein, and chrysophanol (preliminary data), but not alizarin, enhanced transformation of C3H/M2 mouse fibroblasts initiated by N-methyl-N'-nitro-N-nitrosoguanidine or 3-methylcholanthrene. The results of these in vitro studies suggest that hydroxyanthraquinones, possessing 2 hydroxy groups in the 1,8-positions, e.g., danthron, rhein, and chrysophanol, may have tumor-promoting activities. This conclusion is in accordance with the hypothesis that the in vivo carcinogenic activity of danthron may be associated with tumor promotion.

Topics: Animals; Anthraquinones; Carcinogens; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; DNA; Liver; Male; Methylcholanthrene; Methylnitronitrosoguanidine; Rats; Rats, Inbred Strains; Structure-Activity Relationship

Further investigation of the peculiarities of flavin fluorescence photodestruction in malignant cells was made. In normal cells incubated in low pH (3.0-3.2) physiological solutions, the decrease in the oxidized flavoprotein fluorescence intensity under irradiation is the same as in normal pH condition, whereas in tumor cell in low pH solutions a significant increase in the photodestruction level was noticed. The cells isolated from foci of transformation, following treatment of 3T3 NIH fibroblasts with the carcinogen N-methyl-N'-nitro-N-nitrosoguanidine, displayed the photodestruction parameters similar to those in tumor cells. A two-step analysis of cells is proposed for distinguishing between the normal and malignant cells.

Topics: Animals; Cell Line, Transformed; Cell Transformation, Neoplastic; Cells, Cultured; Cytophotometry; Flavins; Fluorescence; Hydrogen-Ion Concentration; Methylnitronitrosoguanidine; Mice; Rats; Tumor Cells, Cultured

Levels of epidermal growth factor (EGF) receptor expression vary widely among cell lines derived clonally from a chemically transformed population of rat liver epithelial cells. Retinoic acid (RA), a derivative of vitamin A that stimulates differentiation in a number of embryonal cell lines, increases the level of 125I-EGF binding in several clones of the transformed cell lines. One such cell line, GP6ac, which reverts to a less transformed phenotype when treated with RA, exhibited a 3-4-fold increase in surface EGF receptors with prolonged (2-5-day) RA exposure. The increase persisted as long as the cells were treated with RA. The increase in surface EGF receptors was due to induction of receptor biosynthesis, which occurred within 4 h at both the mRNA and protein levels and persisted until the RA was withdrawn. Paradoxically, the RA response was accompanied by an initial 40-50% decrease in 125I-EGF binding during the first 12 h of RA treatment. The decrease was due primarily to a reduction of receptor affinity. Since the phorbol ester 12-O-tetradecanoylphorbol-13-acetate also decreases 125I-EGF binding and increases EGF receptor biosynthesis in GP6ac cells, we tested the effect of RA in cells depleted of protein kinase C by prolonged treatment (18 h) with 10 microM 12-O-tetradecanoylphorbol-13-acetate. The absence of protein kinase C did not affect the induction of receptor mRNA and protein or the decrease in binding during the early period of RA exposure. This indicates that RA induction of EGF receptor synthesis in GP6ac cells involves signaling pathways distinct from those utilized by phorbol esters.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Blotting, Northern; Cell Line; Cell Transformation, Neoplastic; Cycloheximide; Epidermal Growth Factor; Epithelial Cells; ErbB Receptors; Gene Expression; In Vitro Techniques; Methylnitronitrosoguanidine; Precipitin Tests; Rats; RNA, Messenger; Tetradecanoylphorbol Acetate; Time Factors; Tretinoin

Four nitropolycyclic aromatic hydrocarbons (NPAHs) were investigated for their cytotoxic effects on rat tracheal epithelial (RTE) cells. 6-Nitrochrysene (6-NC), 1,6-dinitropyrene (1,6-DNP), 1-nitropyrene (1-NP) and 4-nitropyrene (4-NP) induced dose-dependent decreases in the relative colony-forming efficiency (RCFE) of RTE cells. The compounds could be separated into two groups based on their cytotoxic potencies, a group that displayed high cytotoxic effects (6-NC and 1,6-DNP), and a group that displayed low cytotoxic effects (1-NP and 4-NP). The most cytotoxic compound was 6-NC, with an ED50 of 0.13 microM, followed by 1,6-DNP, 4-NP and 1-NP with ED50s of 1.25, 8.9 and 9.1 microM, respectively. The most cytotoxic compound (6-NC) and one of the components with low cytotoxicity (1-NP) were assayed for their ability to induce preneoplastic transformation of RTE cells using equally toxic doses of both compounds. The frequencies of transformation induced by 6-NC in cells isolated from control animals or from animals pretreated with 3-methylcholanthrene (3-MC) were 8.4 X 10(-3) and 21.4 X 10(-3), respectively. 1-NP did not induce cell transformation. Equally toxic doses of the direct acting carcinogen N-methyl-N'-nitro-N-nitrosoguanidine, used as a positive control, induced transformation frequencies of 8.7 X 10(-3) and 6.4 X 10(-3) in cells isolated from control animals or from animals pretreated with 3-MC, respectively. These studies show that RTE cells have the metabolic capacity to activate NPAHs to toxic metabolites; thus, the RTE system should be very useful for evaluating the potential toxic effects of this ubiquitous class of airborne pollutants. In addition, the observed differences in cellular toxicity and transformation capabilities of 6-NC and 1-NP were consistent with the results of other studies that demonstrated the greater potency for induction of tumors in animals of 6-NC relative to 1-NP.

Topics: Animals; Carcinogens; Cell Survival; Cell Transformation, Neoplastic; Cells, Cultured; Chrysenes; Dose-Response Relationship, Drug; Epithelial Cells; Epithelium; Male; Methylnitronitrosoguanidine; Polycyclic Compounds; Pyrenes; Rats; Rats, Inbred F344; Trachea

Primary rat tracheal epithelial cells can be transformed in vitro by N-methyl-N'-nitro-N-nitrosoguanidine. The earliest recognizable morphological transformant is the enhanced growth variant (EGV), characterized by enhanced proliferative capacity. Transformed EGV colonies can progress to give rise to immortal cell lines. The purpose of this study was to determine if specific chromosome changes occur which correlate with immortalization. A total of 34 EGV colonies were isolated, of which five were able to progress in culture to become immortal (greater than or equal to 100 population doublings). Early passages of all five immortalized cultures exhibited additional copies of chromosomes 4, 7, and 11 as a common or recurrent abnormality. These numerical alterations were rarely observed in the primary EGV colonies from which the cell lines were derived, suggesting that these alterations occurred during progression. Structural alterations involving chromosome 1 (resulting in a net gain of 1q) and chromosome 3(3q) also occurred in four out of five immortalized cultures. In all cases, structural alterations involving 1q and/or 3q were detected in the primary EGV colonies from which the immortal cell lines arose. Comparison of the frequency of the structural and numerical alterations observed in the immortalized cultures with their frequency in the 29 EGV colonies which did not become immortal indicated that these changes correlated (P less than or equal to 0.005) with the ability to become immortal. These results suggest that structural alterations occur in primary EGV colonies which predispose cells to immortalization and that subsequent numerical changes occur during progression that correlate with acquisition of the immortal phenotype.

Topics: Aneuploidy; Animals; Cell Transformation, Neoplastic; Chromosome Aberrations; Chromosome Banding; Chromosome Disorders; In Vitro Techniques; Methylnitronitrosoguanidine; Mitotic Index; Rats; Rats, Inbred F344; Trachea

The mouse monoclonal antibody OSU 22-3 was prepared using cells from a squamous cell carcinoma (SCC) as an immunogen. This antibody reacts with an antigen found on squamous cell carcinomas but does not react with normal keratinocytes. This antibody and two antibodies that react with normal keratinocytes were used as markers of malignant and normal phenotypes. These markers were used to evaluate several spontaneous and carcinogen initiated SCC tumors and to identify the expression of an antigen associated with a malignant phenotype. A variety of subpopulations in carcinogen initiated tumors and spontaneous SCC tumors were noted. The subpopulations that reacted only with MoAb OSU 22-3 exhibited features of anchorage independent growth and cellular invasiveness, and formed progressively growing tumors in nude mice. Other SCC spontaneous tumor cell subpopulations reacted with the antibodies associated with normal keratinocytes. These cells did not proliferate in vitro and did not form tumors in the nude mouse. There were other carcinogen transformed cells which reacted with MoAb OSU 22-3 but not with the antibodies associated with normal keratinocytes. These cells exhibited anchorage independent growth and cellular invasiveness but did not form tumors in nude mice. We conclude from this work that human SCC tumors contain multiple cell populations. These cell populations have varied growth properties and express surface antigens that may indicate their malignant vigor. Carcinogen transformed keratinocytes do exhibit some of the characteristics of SCC tumor phenotypes but not the property of malignant progressively growing cells on a routine and consistent basis. This feature is transiently and inconsistently expressed in a surrogate host by populations prepared from spontaneous SSC tumors.

Topics: Animals; Antibodies, Monoclonal; Carcinogens; Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; Dose-Response Relationship, Drug; Epidermal Cells; Epidermis; Humans; Keratins; Lethal Dose 50; Male; Methylnitronitrosoguanidine; Mice; Mice, Nude; Neoplasm Staging; Phenotype; Skin Neoplasms; Tumor Cells, Cultured

Preneoplastic transformants were isolated from primary rat tracheal epithelial cells after treatment with (a) mutagenic concentrations of the alkylating agent N-methyl-N-nitro-N'-nitrosoguanidine, (b) nonmutagenic concentrations of the DNA hypomethylating agent 5-azacytidine, or (c) after arising spontaneously. We have addressed the question of whether preneoplastic transformants induced by different carcinogens differ in their ability to progress to the immortal stage and to become neoplastic. Spontaneous transformants occurred with a very low frequency, and 5-azacytidine induced preneoplastic transformants half as efficiently as N-methyl-N-nitro-N'-nitrosoguanidine. However, no phenotypic differences could be detected between the 70 preneoplastic colonies isolated from the 3 groups; colony size, cell density, and clonogenicity were not statistically different. Clones from all 3 groups became immortal and further progressed to become neoplastic with similar frequencies. The level of expression of the oncogenes H-ras, K-ras, and raf was also similar in all 3 groups. These experiments indicated that there was no difference in the ability of spontaneous transformants or those induced by N-methyl-N-nitro-N'-nitrosoguanidine or 5-azacytidine to progress to become immortal or neoplastic. This suggests that whereas the nature of the carcinogen influenced the frequency of the initial transforming event, progression to the neoplastic stage was independent of the nature of the transforming insult.

Topics: Animals; Azacitidine; Carcinogens; Cell Transformation, Neoplastic; Epithelium; Methylnitronitrosoguanidine; Precancerous Conditions; Rats; Trachea; Tracheal Neoplasms

BALB/MK-2 cells are an epidermal growth factor (EGF)-dependent cell line derived from BALB/c mouse epidermis that can undergo terminal differentiation under appropriate conditions. Previous studies have shown that transformation of these cells by retroviral oncogenes relieves the EGF requirement while blocking the terminal differentiation program. In this report we show that BALB/MK-2 cells are sensitive to transformation by the chemical carcinogens dimethylbenz[a]anthracene (DMBA), 3-methylcholanthrene (MCA), and N-methyl-N'-nitro-N'-nitroso-guanidine (MNNG). BALB/MK-2 cells transformed by these carcinogens proliferate in the absence of EGF and do not undergo terminal differentiation in response to calcium. However, the cells retain their anchorage growth dependence and are nontumorigenic in nude mice. NIH 3T3 transfection analysis showed that the endogenous Ha-ras gene had been activated in both DMBA- and MNNG-transformed cells and the Ki-ras gene had been activated in the MCA-transformed cells. Additionally, non-ras transforming activity was detected in some MNNG-transformed BALB/MK-2 cells. Thus, the BALB/MK-2 cell line provides a reproducible in vitro assay system for chemical transformation of epithelial cells and for identification of oncogene activations associated with changes in growth control and differentiation.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Calcium Chloride; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cells, Cultured; Gene Expression Regulation, Neoplastic; Genes, ras; Keratinocytes; Methylcholanthrene; Methylnitronitrosoguanidine; Mice; Mice, Nude; Nucleic Acid Hybridization; Oncogene Protein p21(ras); Transfection

The MNNG transformed Syrian hamster lung fibroblast cell line (BHLB4) established in our laboratory was treated with 2.0 mmol/L butyric acid. We obtained a butyric acid-dependent partially reversed cell line (ButB4) similar to normal cells in morphology, membrane properties, proliferation rate, colony-forming ability on solid agar culture, etc. However, when transplanted into nude mice, this cell line's tumorigenicity remained intact. Detection of cAMP levels in the cytoplasm showed much higher levels in ButB4 cells than in transformed cells. Butyric acid activity is evidently associated with changes of cAMP. In addition to phenotypical analysis, the karyotypes of ButB4 and BHLB4 were analyzed in detail. It was found that the disomy rate of C15 was significantly increased in ButB4 cells (73%) as compared with BHLB4 cells (33%). These results show that butyric acid not only induces phenotypic reversion in BHLB4 cells directly, but can also selectively kill C15 monosomy cell clones, allowing rapid growth of C15 disomy cell clones. Butyric acid may also influence maintenance of the reversion phenotype.

Topics: Animals; Butyrates; Cell Line, Transformed; Cell Transformation, Neoplastic; Cricetinae; Cyclic AMP; Fibroblasts; Lung; Mesocricetus; Methylnitronitrosoguanidine; Monosomy; Phenotype

The secretion of elevated levels of proteinases is considered to be a distinct property of most transformed cells. The cellular and secreted levels of plasminogen activators and collagenases have been examined in the nonmalignant human osteosarcoma (HOS), the malignant Kirsten murine sarcoma virus transformed (KHOS/NP), the temperature sensitive revertant of virus transformed HOS (KHOS-240S) and N-methyl-N'-nitro-N-nitrosoguanidine transformed HOS (MNNG/HOS) clones. Virus and MNNG transformed clones exhibit 100- and 7-fold higher cellular and and 270- and 30-fold higher extracellular plasminogen activator (PA) activity as compared with untransformed HOS controls. The cellular PA activity of the revertant clone is similar to but the secreted level is slightly higher than the HOS controls. SDS-PAGE in the presence of casein and plasminogen is consistent with the major PA species of urinary type (u-PA) and with the absence of PA inhibitor in the parent and revertant clones. The cellular levels of active collagenase are low in all the clones. However, on activation by trypsin, the two active collagenase bands of similar intensity are observed for all the lines in SDS-PAGE in the presence of gelatin. While there appears to be some elevation of secreted collagenase prior to trypsin activation, the activated collagenases appear to have the same size and activity in all of the clones.

Topics: Cell Line; Cell Transformation, Neoplastic; Electrophoresis, Polyacrylamide Gel; Humans; Kirsten murine sarcoma virus; Methylnitronitrosoguanidine; Microbial Collagenase; Molecular Weight; Plasminogen Activators; Sodium Dodecyl Sulfate

The ability of cultured normal human fetal liver and kidney epithelial cells to repair the premutagenic and precarcinogenic O6-methylguanine (O6-MeGua) DNA adduct was determined by directly monitoring its loss in cellular DNA and quantitating the number of O6-MeGua-DNA-methyltransferase (O6-MT) molecules per cell. Following treatment of the epithelial cells with the direct acting carcinogen N-methyl-N-nitrosourea (MNU), the loss of the O6-MeGua adduct was biphasic, exhibiting a half-life of 2.0 and 1.5 h in the liver and kidney cells, respectively. The activity of O6-MT in the liver and kidney epithelial cells in culture was 0.19 pmol/mg protein or 18,500 molecules/cell. The activity of O6-MT was maintained throughout the life of the cultures, i.e., 20 subpassages or 50 cumulative population doublings for the liver and kidney. In order to ascertain whether human fetal epithelial cells exhibit an induction of O6-MT, the cell cultures were treated with single and multiple conditioning doses of N-methyl-N-nitro-N-nitroso-guanidine (MNNG) or gamma-irradiated and assayed for the amount of O6-MT. A 1 h exposure of cells to 2, 4, and 8 microM MNNG resulted in an 80-100% decrease of the initial O6-MT activity which was restored to the constitutive levels within 48 and 72 h post-treatment. Rat hepatoma cells, used as a positive control, increased their levels of O6-MT to 2.8-fold the constitutive levels following treatment with MNNG. Treatment of the human liver and kidney epithelial cells with chronic low doses of MNNG exhibited O6-MT levels identical to untreated cells. The O6-MT activity in epithelial cells remained unaffected upon pre-irradiation with 1.2 or 2.5 Gy of gamma-irradiation.

Topics: Animals; Carcinogens; Cell Transformation, Neoplastic; Cells, Cultured; DNA Repair; Female; Fetus; Guanine; Humans; Kidney; Liver; Methylnitronitrosoguanidine; Methyltransferases; Pregnancy; Rats

C3H10T1/2CL8 cells treated on the first day after seeding with benzo[a]pyrene (B[a]P) and then treated again with B[a]P displayed an inhibited response of morphological transformation if the second treatment was administered from 14 days to 33 days after seeding. Under these conditions the cells exhibited up to 100% inhibition of morphological transformation, the extent of inhibition being related to the concentration of B[a]P administered in the second treatment. 3-Methylcholanthrene (3MC) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) also inhibited B[a]P-induced morphological transformation as a function of concentration when administered to cells 21 days after the initial B[a]P treatment. Delayed recovery of transformed foci was examined in cells treated with B[a]P on days 1 and 22 and scored 6-9 weeks after the first B[a]P treatment. No recovery of cell transformants was observed. Reconstruction experiments with normal and transformed C3H10T1/2CL8 cells suggested that selective cytotoxicity to incipient transformed cells could account for the inhibition by MNNG, but could not account for up to 50% of the inhibition induced by the second treatment of B[a]P or 3MC.

Topics: Animals; Benzo(a)pyrene; Carcinogens; Cell Line; Cell Transformation, Neoplastic; Chromosome Aberrations; Dose-Response Relationship, Drug; Methylcholanthrene; Methylnitronitrosoguanidine; Mice; Mice, Inbred C3H

The abilities of various dioxins to induce toxicity or transformation of rat tracheal epithelial cells in culture were examined. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) was not cytotoxic and did not induce transformation as measured by the induction of growth-altered, preneoplastic cells (termed enhanced growth variants). However, TCDD enhanced the transformation of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-initiated rat tracheal epithelial cells. Other dioxin congeners with 0 to 3 chloro substitutions were inactive in enhancing MNNG transformation. TCDD was most effective at a concentration of 0.3 nM and when treatment was administered immediately after MNNG exposure. The dose-response curves for enhancement of MNNG-induced transformation and induction of aryl hydrocarbon hydroxylase activity by TCDD were similar. These results are consistent with the hypothesis that the enhancement of cell transformation by TCDD is mediated through the TCDD receptor. TCDD also enhanced transformation when the cells were treated before MNNG treatment. The ability of 12-O-tetradecanoylphorbol-13-acetate (TPA) to enhance MNNG-induced rat tracheal epithelial transformation was also examined. In contrast to the findings with TCDD, the number of transformed colonies was increased only by pretreatment with TPA followed by MNNG. TPA-pretreatment enhanced equally the number of normal cells forming colonies and the total number of transformed colonies after selection; therefore, the transformation frequency (transformants per total surviving colonies) was unchanged by TPA. In contrast, TCDD treatment enhanced the transformation frequency in MNNG-exposed cultures since the number of transformed colonies increased while the number of total colonies remained constant. Thus, TCDD appears to act by a different mechanism than TPA. TCDD enhancement of MNNG-induced transformation may be attributed to a promotional effect, a comutagenic action, or a modulation of cell proliferation and/or differentiation mediated through the TCDD receptor.

Topics: Animals; Cell Transformation, Neoplastic; Cells, Cultured; Dioxins; Dose-Response Relationship, Drug; Epithelium; Male; Methylnitronitrosoguanidine; Polychlorinated Dibenzodioxins; Rats; Rats, Inbred F344; Tetradecanoylphorbol Acetate; Trachea

Transforming growth factor beta (TGF beta) is an inhibitor of normal epithelial cell growth. To investigate the role of TGF beta in respiratory epithelial cell neoplasia, normal, preneoplastic, tumorigenic and tumor-derived rat tracheal epithelial (RTE) cells were plated in serum-free medium and grown in the presence of 0-300 pg TGF beta 1/ml. TGF beta 1 markedly inhibited the formation of colonies by primary RTE cells and some preneoplastic RTE cells. However, tumor-derived RTE cells were relatively resistant to TGF beta 1-induced growth inhibition. Resistance to TGF beta 1-induced growth inhibition, therefore, accompanies neoplastic progression of RTE cells.

Topics: Animals; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cells, Cultured; Drug Resistance; Epithelial Cells; Epithelium; Male; Methylnitronitrosoguanidine; Mice; Mice, Nude; Precancerous Conditions; Rats; Rats, Inbred F344; Trachea; Transforming Growth Factors

Many studies have shown that cultured rat liver epithelial cells can be neoplastically transformed by repeated or long-continued exposure to chemical carcinogens. These cells also may transform spontaneously in the absence of carcinogen treatment after long-term, continuous passage in culture or after chronic maintenance in a confluent state in vitro. In this study, we have compared the times of emergence and rates of accumulation of transformed cells in populations of rat hepatic epithelial cells exposed either to a single dose of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG, 3 micrograms/ml culture medium for 30 minutes) or to acetone vehicle alone (3 microliters/ml culture medium for 30 minutes). Transformation was compared in cell populations that were passaged continuously once a week as they attained a confluent density (nonselective growth conditions), or that were maintained at a confluent density for 3 weeks between passages once a month (selective growth conditions). Emergence of both spontaneous transformants and transformants induced by MNNG was facilitated by selective growth conditions, as compared with non-selective growth conditions. Transformants were detected both in cultures exposed to MNNG and in cultures exposed only to acetone (solvent controls), but they always emerged earlier in cultures exposed to MNNG (nine population doublings earlier when grown under selective growth conditions and 22 population doublings earlier when grown under nonselective growth conditions). Once transformants were detected, they replaced the nontransformed population more quickly under selective than under nonselective conditions of culture. Cells possessing the ability to grow in soft agar and to produce tumors in syngeneic rats were detected (at about 12 population doublings after treatment) under selective conditions much earlier than under nonselective growth conditions (at about 90 population doublings after treatment). Among MNNG-treated cultures, the fraction of aneuploid cells in the population was correlated significantly with tumorigenicity. In contrast, among acetone-treated control populations, aneuploidy and tumorigenicity were not correlated; populations of aneuploid acetone-treated cells often were not tumorigenic. These observations suggest that MNNG treatment produced a specific type of aneuploidy that was associated with tumorigenicity.

Topics: Animals; Carcinogenicity Tests; Cell Transformation, Neoplastic; Cells, Cultured; Epithelium; Flow Cytometry; Karyotyping; Liver; Male; Methylnitronitrosoguanidine; Rats; Rats, Inbred F344

The present studies intend to heighten the sensitivity of BALB/3T3 cells to chemical carcinogens in a transformation assay, by including exposure of carcinogen-treated cells to a tumor promoter, 12-O-tetradecanoylphorbol 13-acetate (TPA). In the assay, cells were first treated with a known or suspected carcinogen for 72 h, cultured in normal medium for 3 days, exposed to media with and without TPA for 2 weeks, and cultured in normal medium for an additional 3 weeks. Benzo[a]pyrene, a potent carcinogen with a polycyclic aromatic hydrocarbon structure, caused transformation in the presence and absence of TPA. N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG), a carcinogen with direct-acting alkylating ability, did not induce significant transformation without TPA, while treatment with MNNG followed by TPA produced numerous transformed foci, classifying MNNG as an initiating agent of transformation under the condition presented in this report. 3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2), sodium nitrite and butylated hydroxyanisole (BHA), which are carcinogenic and/or mutagenic, produced transformed foci in significant numbers of treated dishes in the presence but not in the absence of TPA. Butylated hydroxytoluene (BHT) and sodium saccharin, which are considered to be a modifier and a promoter of carcinogenesis, did not cause significant transformation with or without TPA treatment. These studies suggest that this 2-stage transformation system is capable of detecting a wider range of chemical carcinogens as initiating agents than the standard assay. Studies on the transformation assay schedule revealed that the proportion of dishes with foci, the number of foci per dish and sizes of foci all increased in the normal medium after the termination of TPA treatment. Therefore, transformed cells appear to proliferate independently of TPA after those cells are released by TPA from postconfluence inhibition of cell division.

Topics: Animals; Carcinogens; Cell Transformation, Neoplastic; Cells, Cultured; Methylnitronitrosoguanidine; Mice; Mice, Inbred BALB C; Mutagens; Tetradecanoylphorbol Acetate

Diminished intercellular communication has been associated with heightened sensitivity of cultured cells to morphological transformation and enhancement of transformation by tumor promoters. Microinjection of Lucifer yellow dye was employed to evaluate intercellular communication between transformable C3H/10T1/2 murine fibroblasts under a variety of culture conditions. Intercellular communication assayed by dye transfer from injected cells to surrounding cells in contact occurred in logarithmically growing cultures, declined to very low levels as confluence was attained, and then resumed upon the formation of mature confluent monolayers. Dye-transfer networks of 50 or more cells resulted from injection of single monolayer cells. Freeze-fracture electron microscopy confirmed the presence of gap junction structures in confluent cultures. Treatment with the initiating agent N-methyl-N'-nitro-N-nitrosoguanidine and/or the tumor promoter 2,3,7,8-tetrachlorodibenzo-p-dioxin did not inhibit intercellular communication between C3H/10T1/2 cells during 6-week transformation experiments. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate produced a transient inhibition of dye-coupling upon first introduction to cultures and prolonged the period of diminished dye-coupling at the attainment of confluence, but did not inhibit subsequent interactions between monolayer cells. A simple relationship thus could not be established between levels of dye-coupling within monolayers and focus formation events. Curiously, although the cells of foci in early phases of development did not exhibit dye-transfer capacity, dye-coupling was observed in mass cultures of most transformed cell lines cloned from foci. Co-cultivation of communication-competent transformed cells with nontransformed cells to produce reconstructed foci generally resulted in a cessation of dye-transfer by transformed cells. An often reversible loss of communication competence thus accompanies the growth of transformed C3H/10T1/2 cells as foci and may constitute an adaptive response which facilitates focus growth in the presence of intercellular communication between monolayer cells.

Topics: Animals; Carcinogens; Cell Communication; Cell Line; Cell Transformation, Neoplastic; Isoquinolines; Methylcholanthrene; Methylnitronitrosoguanidine; Mice; Polychlorinated Dibenzodioxins; Tetradecanoylphorbol Acetate

To determine the role of repair of potentially lethal damage (PLD) in the initiation process of neoplastic transformation, Balb/c 3T3 cells treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were temporarily exposed to conditioned medium obtained from density-inhibited Chinese hamster cell cultures, as a post-treatment for the induction of PLD repair. With or without this exposure, cell survival and transformation frequencies were simultaneously determined by colony-formation and focus-formation assays, respectively. Temporary exposure to conditioned medium resulted in a 20-30% increase in cell survival compared with no exposure. Post-treatment with conditioned medium resulted in a 60-65% reduction in transformation frequencies. At the molecular level, the repair of MNNG-induced single-strand breaks of DNA occurred much more rapidly in conditioned medium. These data suggest that PLD repair reduces the in vitro neoplastic transformation through excision repair operative during the cessation of DNA replication. Thus, PLD repair appears to be preventive against neoplastic fixation in initiation of neoplastic development.

Topics: Animals; Cell Survival; Cell Transformation, Neoplastic; Cells, Cultured; DNA Repair; DNA Replication; Methylnitronitrosoguanidine; Mice; Mice, Inbred BALB C

An in vivo-in vitro approach to studying neoplastic development in carcinogen-exposed rat fibroblasts was evaluated. In the model described, oncomodulin (Mr 12,000; pI 3.9), a tumor-associated and Ca2+-binding protein, was used as a specific marker of malignant transformation. A rapidly proliferating granulation tissue was exposed in vivo or in vitro to potent carcinogens like N-methyl-N'-nitro-N-nitrosoguanidine and procarbazine. As an endpoint of transformation anchorage independent (AI) colony formation in the soft agar assay was chosen. Exposure to various doses of N-methyl-N'-nitro-N-nitrosoguanidine in vivo or in vitro, or to procarbazine in vivo, led to induction of AI, transformed cells. Exposure of the cells to various doses of procarbazine in vitro produced neither formation of AI cells in the agar nor expression of oncomodulin in extracts of the exposed cell population. Almost all of the chemically induced AI cell lines tested have been found to be tumorigenic in athymic mice. In contrast, a very low rate (zero to two colonies per 10(6) cells tested) of spontaneous AI populations derived from untreated cells. None of these control AI colonies yielded tumors. In our transformation assay the appearance of neoplastic phenotypes seems very rapid, probably due to the increased cell division at the time of carcinogen-exposure. Expression of oncomodulin was found in extracts of transformed cells harvested from agar colonies, derived from carcinogen-exposed granulation tissue, but not from normal, untreated fibroblasts, as shown by two-dimensional polyacrylamide gel electrophoresis and high-performance liquid chromatographic analysis, as well as 45Ca2+-transblot electrophoresis. The presence of oncomodulin in extracts of transformed cells correlates well with the chemically induced colony formation in the soft agar assay. Oncomodulin might be a suitable neoplastic marker to study chemical carcinogenesis.

Topics: Animals; Biomarkers, Tumor; Calcium; Calcium-Binding Proteins; Cell Survival; Cell Transformation, Neoplastic; DNA Replication; Fibroblasts; Granuloma; Kinetics; Male; Methylnitronitrosoguanidine; Neoplasm Proteins; Rats; Rats, Inbred Strains

Following carcinogen treatment, elevated expression levels of dihydrofolate reductase (dhfr) were measured by labeling cells with fluorescent methotrexate which binds to this enzyme. Fractionation of carcinogen-treated, Simian virus 40 (SV40)-transformed Chinese hamster embryo cells (C060) into subpopulations differing in their levels of dhfr expression revealed co-expression, at enhanced levels, of dhfr and SV40 T antigen in the same cells. The increased expression of dhfr was amplification independent, while the T antigen coding sequences were amplified. The co-expression of dhfr and the bacterial chloramphenicol acetyltransferase gene linked to the early SV40 regulatory region was measured in CHO cells stably transformed by pSV2CAT-SVgpt (CC24). Both these sequences were expressed at higher levels in treated cells and the elevated expression levels were observed in the same subpopulation of cells, although no increase in their gene copy number was detected. The concomitant activation of enhanced expression of two independent genes in the same cells suggests that cellular factors governing gene expression are activated in the carcinogen-treated cells. The implications of these findings to cellular control mechanisms and to the carcinogenic process are discussed.

Topics: Animals; Antigens, Polyomavirus Transforming; Cell Line, Transformed; Cell Transformation, Neoplastic; Cricetinae; Cricetulus; Gene Expression Regulation; Genes; Genes, Viral; Methylnitronitrosoguanidine; Plasmids; Simian virus 40; Tetrahydrofolate Dehydrogenase

Carcinogenesis is considered to be a multi-step process comprising 'initiation', 'promotion' and 'conversion' events. Skin fibroblasts from patients with hereditary retinoblastoma (RB) and familial polyposis coli (FPC) were chosen for study since their predisposition to the tumour may be due to an inherited 'initiation' event which is present in every cell. Experiments involving skin fibroblasts from FPC patients showed certain of these cells to grow in semi-solid medium following treatment with the complete carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) alone. When tested prior to the commencement of the experiments the FPC patient cell populations had shown no strong predisposition to malignant transformation as assessed by increased saturation densities, reduced serum requirements, altered migration patterns in collagen gels, anchorage-independent growth and tumourigenicity in nude mice. Following carcinogen or promoter treatment, apart from exhibiting low-level frequencies of anchorage-independent growth, the cells appeared no more transformed than they were before. Parallel cytogenetic studies showed TPA to increase both tetraploidy and the chromosome-aberration frequency during the course of these transformation studies. However, the FPC cell clones induced by TPA to grow in semi-solid medium were, at best, considered to be only partially transformed when their properties were compared with those of tumour-derived cell lines.

Topics: Adenomatous Polyposis Coli; Cell Adhesion; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Chromosome Aberrations; Eye Neoplasms; Female; Fibroblasts; Humans; Male; Methylnitronitrosoguanidine; Ploidies; Retinoblastoma; Skin

Treatment of skin fibroblasts from an FPC patient with 4NQO or MNNG followed by sequential passaging caused morphological changes of the cells, which showed characteristics of transformed cells such as a high frequency of colony formation in agarose, increased growth ability, and chromosomal abnormalities. This and other fibroblast lines from 5 of 12 FPC patients had an increased susceptibility to 4NQO cytotoxicity, which was caused by enhanced 4NQO-reductase activity rather than by reduced DNA repair. However, the susceptibility to cytotoxicity of MNNG and repair of MNNG-damaged DNA were normal in FPC cells. The tumor promoters TPA and DHTB enhanced the frequency of chemical transformation of the FPC fibroblasts, and protease inhibitors suppressed the promoter-enhanced transformation. The skin fibroblasts from many FPC patients exhibited increased susceptibility to transformation by murine sarcoma viruses. Analysis of the viral DNA and RNA after infection revealed that the increased susceptibility is determined at an early stage of transformation. Two out of 5 MNNG-transformed clones of FPC fibroblasts, isolated from agarose, had increased expression of c-Ki-ras or c-Ha-ras, and 4 of 4 MSV-transformed clones showed high expression of viral Ki-ras. These clones grew further after isolation from agarose, but were mortal and did not form tumors in nude mice. The present results suggest that additional changes in morphologically transformed FPC fibroblasts are required for malignant transformation.

Topics: 4-Nitroquinoline-1-oxide; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Chromosome Aberrations; Chromosome Disorders; Fibroblasts; Humans; Karyotyping; Methylnitronitrosoguanidine; Mitosis; Moloney murine sarcoma virus; Nitroquinolines; Sarcoma Viruses, Murine; Skin

Exposure of synchronized C3H10T1/2 (clone 8) cell populations of various sizes to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) at a concentration of 2 micrograms/ml for 30 min at 24 h after release from confluence-induced arrest of proliferation produced neoplastic transformation (formation of foci of morphologically altered cells) by a random but episodic process in a small fraction of the cells at risk soon after treatment. The fraction of dishes that contained type II or type III foci increased as the number of cells at risk increased. In contrast, the development of spontaneous foci is a stochastic process that depends on the number of new cells that form during population growth and is independent of the number of cells that are plated (J. W. Grisham et al., Cancer Res., 48: 5969-5976,1988). When there were small numbers of cells at risk, spontaneous formation of foci was a source of considerable error in evaluating MNNG-induced transformation frequency. In surviving cell populations of less than 1000-3000 cells/100-mm dish, the frequency of induction of foci by MNNG could not be distinguished statistically from the frequency with which foci were expected to form spontaneously. When the fraction of MNNG-treated dishes that contained foci was adjusted for the fraction of pooled control dishes that contained foci, the number of foci induced by a uniform dose of MNNG was found to vary with the number of surviving cells. However, the MNNG-induced transformation frequencies calculated by the Poisson method were independent of the size of the population of cells at risk, provided the population of cells at risk was of sufficient size to allow spontaneous and induced transformation to be distinguished statistically. The results of this study show that the frequency of MNNG-induced transformation can be quantitated in cultures of 10T1/2 cells that contain varying but sufficient numbers of cells at risk when spontaneous transformation is considered. Furthermore, these observations suggest that MNNG-induced transformation of 10T1/2 cells occurs with the frequency and characteristics of a mutation-like change involving a single gene.

Topics: Animals; Cell Transformation, Neoplastic; Cells, Cultured; Methylnitronitrosoguanidine; Mice; Mutation; Proto-Oncogenes; Sodium-Potassium-Exchanging ATPase; Tetradecanoylphorbol Acetate

We compared proliferation and survival of various syngeneic transformed cell lines under conditions of depletion of 15 amino acids in Dulbecco-Eagle's medium. We used a normal fibroblast line 3Y1 and 22 transformed sublines of 3Y1 which had been induced by one of seven transforming agents--simian virus 40, mouse polyomavirus, adenovirus type 12, E1A gene of adenovirus type 12, cDNA of Harvey murine sarcoma virus, Rous sarcoma virus, or N-methyl-N'-nitro-N-nitrosoguanidine. Unlike other untransformed cells examined (mouse BALB/c-3T3 line, mouse NIH-3T3 line, and primary Fischer rat embryo fibroblasts), 3Y1 ceased to proliferate and accumulated in a viable state with a G1-phase DNA content under 14 singular deprivations of amino acid. None of the transformed 3Y1 lines completely arrested in the G1 phase of the cell cycle and each showed different levels of survival, depending on each transforming agent. As for transformed 3Y1 cells induced by a given virus or a given transforming gene, any one of the three sublines shared the same trend with respect to proliferation and survival. Transformed derivatives induced by N-methyl-N'-nitro-N-nitrosoguanidine showed almost the same trend in proliferation, but the patterns of survival were not uniform. Our observations suggest that the unique responses of 3Y1 to amino acid depletion are differently modified by different transforming agents.

Topics: Adenoviridae; Amino Acids; Animals; Cell Division; Cell Line; Cell Survival; Cell Transformation, Neoplastic; Cell Transformation, Viral; DNA; Fibroblasts; Methylnitronitrosoguanidine; Mice; Mice, Inbred BALB C; Polyomavirus; Rats; Rats, Inbred F344; Simian virus 40

Transformation experiments have been carried out on human diploid fibroblasts derived from normal individuals and those from 2 groups with dominantly inherited cancer predisposition, familial polyposis coli (FPC), and multiple endocrine neoplasia, type 2 (MEN-2). Treatment with a single or multiple doses of the carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), resulted in limited anchorage-independent (AI) growth in both normal and FPC cultures; no permanent cell lines were produced but FPC cells showed increased proliferation with low doses of the carcinogen. Carcinogen treatment followed by application of the tumour promoter, 12-O-tetradecanoyl phorbol-13-acetate (TPA), for 38 weeks was insufficient to cause full transformation in cultures derived from normal people or MEN-2 patients although AI growth was induced in all 3 cell types. Three FPC cultures exhibited an extended life span over the solvent controls. Two of these are still actively dividing and have a clonal pseudodiploid karyotype.

Topics: Adenomatous Polyposis Coli; Cell Division; Cell Transformation, Neoplastic; Fibroblasts; Humans; Kinetics; Methylnitronitrosoguanidine; Multiple Endocrine Neoplasia; Neoplasms; Skin; Tetradecanoylphorbol Acetate

Spontaneous formation of morphologically altered foci of types II and III (neoplastic transformation) was examined in populations of C3H 10T1/2 (10T1/2) cells. Initial surviving cell densities ranged from 3 to 3 x 10(5) cells/100-mm dish and the final cell density was approximately 2 x 10(6) cells/dish, yielding widely differing numbers of population doublings but similar numbers of cell births from the time of cell plating to the attainment of confluence. Spontaneous formation of foci was independent of the initial surviving cell densities (and, therefore, of the number of population doublings) but was related to the number of cell divisions (cell births) between the time the cell population was plated and when suppression of proliferation of wild-type cells occurred in confluent cultures. In 418 pooled asynchronously proliferating cultures in 100-mm dishes the 95% confidence limits for the fraction of dishes containing foci was 0.041-0.089 for type II foci and 0.008-0.036 for type III foci; for cell populations in 2041 pooled cultures in 100-mm dishes, the proliferation of which was synchronized by release from confluence-induced arrest of proliferation, the 95% confidence limits for the fraction of dishes containing foci were 0.150-0.166 for type II foci and 0.017-0.032 for type III foci. Using the Poisson method, the 95% confidence limits for rates of spontaneous transformation in asynchronously proliferating populations of 10T1/2 cells were 1.4-3.2 x 10(-8)/cell/division for type II foci and 0.28 to 1.3 x 10(-8)/cell/division for type III foci; in populations in which proliferation was initially synchronized by release from confluence-induced arrest, spontaneous transformation rates were 5.6-6.3 x 10(-8)/cell/division for type II foci and 0.59-1.1 x 10(-8)/cell/division for type III foci. Spontaneous transformation occurred in populations of wild-type 10T1/2 cells at the rates and with the characteristics expected of the mutation of a single gene locus.

Topics: Animals; Cell Communication; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Methylnitronitrosoguanidine; Mice; Mutation; Phenotype

We have investigated the activity of insoluble and soluble lead compounds in inducing mutagenesis, cell transformation and sister chromatid exchange in mammalian cells. Insoluble lead sulfide, readily phagocytized, was more than four times as toxic to V79 cells on a microM basis, than two moderately soluble lead compounds although the exposure time for the soluble salts was five times longer. These findings demonstrate the importance of different cellular mechanism(s) of metal uptake and bioavailability. Both insoluble lead sulfide and more soluble lead nitrate were mutagenic at the HPRT locus in V79 cells. Although less mutagenic at the higher concentrations, lead nitrate at a concentration of 500 microM enhanced the mutation frequency greater than 6-fold above background following a 5-day exposure. Although the mechanism(s) by which lead induces mutations is unknown, failure of both compounds to induce SCE and DNA single-strand breaks, detectable by alkaline elution, suggests that lead-induced mutations may not be a result of direct damage to DNA but may occur via indirect mechanisms including disturbances in enzyme functions important in DNA synthesis and/or repair, or in DNA-helical structure. Lead acetate also transformed SHE cells in a dose-response fashion following a 48-h exposure. Our results indicate that lead compounds may be genotoxic by an indirect mechanism, and lend support to the view that lead is a carcinogen.

Topics: Animals; Carcinogens; Cell Line; Cell Transformation, Neoplastic; Hypoxanthine Phosphoribosyltransferase; Lead; Methylnitronitrosoguanidine; Mutagenicity Tests; Mutagens; Phagocytosis; Sister Chromatid Exchange

2,2-Dichlorovinyl-O,O-dimethyl phosphate (DDVP), an extensively used household insecticide, was assayed for its genotoxicity in primary rat tracheal epithelial (RTE) cells. Cytotoxicity of DDVP to RTE cells was dose-dependent, killing about 50% of the cell population at a dose of 80 micrograms/ml. Sister-chromatid exchange (SCE) and chromosomal aberrations induced by this insecticide were positive in RTE cells although the doses needed for significant inductions were much higher than those by a known genotoxic agent, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The level of transformation induced by DDVP in RTE cells was about 1/5 that induced by MNNG at a dose of similar cytotoxicity. The slope of the regression line for induced transformation is 1.27. DDVP probably induces the genotoxic effect in RTE cells by a one-hit mechanism.

Topics: Animals; Cell Survival; Cell Transformation, Neoplastic; Cells, Cultured; Chromosome Aberrations; Dichlorvos; Epithelium; Male; Methylnitronitrosoguanidine; Mutagenicity Tests; Rats; Rats, Inbred Strains; Sister Chromatid Exchange; Trachea

The temporal order of replication of several genes was studied in 10T1/2 cells synchronized by release from confluence-induced arrest of proliferation followed by treatment with 2 micrograms/mL aphidicolin for 24 h. DNA subjected to bromodeoxyuridine substitution for 1- or 2-h intervals spanning the S phase was separated from the remaining DNA in cesium chloride gradients, filtered onto nitrocellulose in a slot-blot apparatus, and hybridized with various 32P-labeled probes. Ha-ras was among the first genes replicated at the onset of the S phase. The myc proto-oncogene replicated later but within the first hour of the S phase. The replication of Ki-ras, raf, and mos was detected between hour 1 and 2 of the S phase. The dihydrofolate reductase gene replicated early (0-2 h) and the myb proto-oncogene replicated in mid-S phase (2-4 h). An immunoglobulin VH sequence and the beta-globin gene replicated late in 10T1/2 cells, 4-6 h after removal of aphidicolin. Replicating DNA is preferentially adducted by chemical carcinogens, and replication of damaged proto-oncogenes before they are repaired may activate their transforming potential. Therefore, the observed replication of proto-oncogenes during the early S phase may underlie the enhanced sensitivity of 10T1/2 cells to chemically induced transformation at this point in the cell cycle.

Topics: Animals; Autoradiography; Cell Transformation, Neoplastic; Cells, Cultured; DNA Probes; DNA Replication; Immunoblotting; Interphase; Methylnitronitrosoguanidine; Mutation; Nucleic Acid Hybridization; Plasmids; Proto-Oncogenes; Time Factors

The secretion of transforming growth factors (TGFs) alpha and beta by normal, chemically transformed, and malignant rat liver epithelial cell lines was investigated. The WB-F344 normal cultured rat liver epithelial cell line does not secrete an epidermal growth factor-like (putatively TGF-alpha) activity, but several clonal cell strains derived from WB-F344 cells which had been treated with N-methyl-N'-nitro-N-nitrosoguanidine, especially those that expressed high levels of gamma-glutamyl transpeptidase, secreted TGF-alpha-like activity into their conditioned media. Cell lines obtained from tumors which were produced by these cell strains varied in their abilities to secrete TGF-alpha, even though they all expressed high levels of gamma-glutamyl transpeptidase activity. When two of the non-TGF-alpha-secreting tumor cell lines were transplanted into isogeneic rats, the tumors that formed contained high levels of TGF-alpha-like activity. Although epidermal growth-factor (hence, TGF-alpha also) inhibited the proliferation of several of these tumor cell lines in monolayer cultures, this growth factor often paradoxically stimulated the anchorage-independent growth of the same cell lines. In contrast to TGF-alpha-like activity, all cell lines/strains released TGF-beta activity into their conditioned media. However, while both normal or chemically transformed cell strains typically produced the inactive form of TGF-beta, the tumor cell lines tended to produce activated TGF-beta de novo. Anchorage-independent growth of cell lines that produced active TGF-beta was either stimulated, inhibited, or unaffected by TGF-beta. Cell lines that were inhibited by TGF-beta concurrently produced TGF-alpha which was usually able to overcome the negative "autocrine" effect of TGF-beta. We conclude that both TGF-alpha and TGF-beta, singly or in combination, are variously involved in the growth of transformed rat liver epithelial cells. TGF-alpha has a predominantly positive autocrine action on the growth of rat liver epithelial tumor cell lines. The "paracrine" effect of TGF-beta may be at least as important as its autocrine effect in the growth of these transformed epithelial cell lines.

Topics: Animals; Cell Division; Cell Line; Cell Transformation, Neoplastic; Epithelial Cells; Epithelium; ErbB Receptors; Growth Substances; Insulin; Liver; Methylnitronitrosoguanidine; Neoplasm Proteins; Peptide Biosynthesis; Peptides; Rats; Transforming Growth Factors

A comparative analysis of T24 human bladder carcinoma cells and N-methyl-N'-nitro-N-nitrosoguanidine (MeNNG)-transformed derivatives (MeNNG-T24 cells) revealed the following: (i) The presence of an activated c-Ha-ras gene (in the absence of the normal allele) is insufficient to confer upon T24 cells a tumor-associated phenotype. (ii) MeNNG-transformed T24 cells not only acquire tumor-associated (in vitro) traits (growth in soft agar and rhodamine retention) but, are highly tumorigenic in nude mice. (iii) It is possible to render T24 cells tumorigenic by chemical transformation; therefore, the reason that T24 cells lack tumorigenicity is not because of possible incompatibilities between these cells and nude mice but, in fact, because T24 cells are not malignant. (iv) The loss of expression of a transformation-related Mr 67,000 phosphoprotein by MeNNG-T24 cells after explantation of these cells from nude mouse tumors to in vitro culture indicates that culture conditions can be responsible for rapid phenotypic conversion of human tumor cell lines.

Topics: Animals; Cell Transformation, Neoplastic; Humans; Methylnitronitrosoguanidine; Mice; Mice, Nude; Molecular Weight; Neoplasm Transplantation; Nucleic Acid Hybridization; Phenotype; Phosphoproteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); Proto-Oncogenes; RNA, Messenger; Tumor Cells, Cultured; Urinary Bladder Neoplasms

The cytogenetic changes in enhanced growth (EG) variants of rat tracheal epithelial cells in culture were examined. These variants which are detectable at 35 days after carcinogen exposure are the first phenotypic alteration in the multistep neoplastic process studied in this model system. Karyotypic analysis of N-methyl-N'-nitro-N-nitrosoguanidine-induced EG variants at Day 35 was made possible by the development of an in situ method of cytogenetic analysis on intact colonies containing too few cells for conventional chromosome preparation methods. Of the transformed EG variant colonies in both control and N-methyl-N'-nitro-N-nitrosoguanidine-treated groups, 62-78% had abnormal karyotypes which included numerical and structural changes. There were no specific chromosome changes, although aberrations of chromosomes 3 and 4 were recurrently observed. However, some colonies of even the most morphologically transformed EG variants were composed of only diploid cells. To confirm this finding 10 EG variant colonies were bisected and half of the clone was prepared for chromosome analysis and the other half was subcultured to measure the clonogenicity and karyotypes of the cells. Cells from 3 colonies plated very poorly on 3T3 feeders and therefore no karyotypic analysis of the colony-forming cells was possible; the cells of the 3 parental colonies were diploid. Three other parental colonies were predominantly diploid (80-90%) but upon replating the resultant daughter colonies had progressively smaller fractions of diploid cells indicating a selection for cells with abnormal karyotypes. When more selective conditions were used (i.e., growth after removal of the feeder cells), the percentage of abnormal cells increased even further. In one case the parental cells had a karyotypic alteration in the long arm of chromosome 4 and this karyotypic alteration was accentuated in the daughter colonies. Thus, selection of cells with increased growth ability upon subculturing or growth in the absence of feeder cells (properties associated with the acquisition of immortality) resulted in concomitant selection for cells with abnormal karyotypes. Since some of the carcinogen-induced rat tracheal epithelial cells expressing the EG variant phenotype were diploid, it is possible that the first step in this transformation process is an epigenetic change. However, most of the diploid cells became terminal. The aneuploid subpopulations present in these colonies have a selective grow

Topics: Aneuploidy; Animals; Cell Transformation, Neoplastic; Chromosome Aberrations; Chromosome Banding; Epithelium; In Vitro Techniques; Karyotyping; Methylnitronitrosoguanidine; Neoplastic Stem Cells; Precancerous Conditions; Rats; Time Factors; Trachea; Tumor Cells, Cultured

Topics: Animals; Calcium; Calcium-Binding Proteins; Cell Transformation, Neoplastic; Methylnitronitrosoguanidine

The strong carcinogens, N-methyl-N-nitrosourea (MNU), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) react with phosphatidylethanolamine (PE) in chicken erythrocyte ghosts or rat kidney cells, whereas a weak carcinogen, methyl methanesulphonate (MMS), does not. The reactions of these agents with commercial PE were also carried out and their reactivities were compared with their carcinogenic potencies. When compared with the amount of PE of the blank, 64%, 18% and 0% of PE were reacted with MNNG, MNU and MMS respectively. This indicated that MNNG is approximately 4 times more reactive than MNU, while MMS does not react with PE, correlating with their tumorigenic potencies on mouse skin. A positive correlation was also observed between the reactivity of a series of N-methyl-N'-aryl-N-nitrosoureas (I-X) to PE and their tumorigenicity. In the case of the nitrosoureas, the reactions proceeded through carbamoylation of the amino group in PE by the isocyanates generated from the agents. Furthermore, many isocyanates also reacted with PE just as the strong carcinogens did. Our present results suggest that both alkylation of DNA and reaction with cell membranes would be required for the formation of transformed benign cells. For progression of the benign to malignant cells, further alkylation of DNA would be required, and the carcinogenic potency of the agents may result from a combination of both reactions.

Topics: Animals; Cell Membrane; Cell Transformation, Neoplastic; Chickens; Erythrocyte Membrane; Kidney; Magnetic Resonance Spectroscopy; Mass Spectrometry; Membrane Lipids; Methyl Methanesulfonate; Methylnitronitrosoguanidine; Methylnitrosourea; Phosphatidylethanolamines; Rats

BALB/c 3T3 and its derivative MO-5, isolated as a monensin-resistant clone, showed a very low rate of spontaneous malignant transformation. Treatment of BALB/c 3T3 cells with benzo(alpha)pyrene, 4-nitroquinoline-N-oxide, N-methyl-N'-nitro-N-nitrosoguanidine, N-methyl-N-nitrosourea, or UV light irradiation significantly enhanced the rate of transformation, whereas the treatment of MO-5 cells with these carcinogens had only a slight if any effect. Exposure of MO-5 as well as BALB/c 3T3 to 5 or 10 microM 5-azacytidine for 3 to 7 days significantly increased the number of transformation foci. The Luria-Delbrück fluctuation test showed that spontaneous mutation frequency (mutants/cell/generation) was 1.2 X 10(-6) for BALB/c 3T3 and 7.1 X 10(-7) for MO-5, respectively, when appearance of cadmium-resistant clones was tested. N-Methyl-N'-nitro-N-nitrosoguanidine enhanced induced mutation frequency of ouabain-resistant and cadmium-resistant mutants of BALB/3T3 but it only slightly enhanced that of MO-5. Methylation status of DNA of MO-5 was compared with that of BALB/c 3T3 by comparing the cleavage patterns generated by the isoschizomeric restriction enzymes HpaII and MspI. DNA of MO-5 was found to be more methylated than that of BALB/c 3T3 in the vicinity of c-myc as well as the metallothionein-I gene. Aberrant DNA methylation in MO-5 and the cellular sensitivity to transformation by chemical carcinogens or 5-azacytidine are discussed.

Topics: 4-Nitroquinoline-1-oxide; Animals; Azacitidine; Benzo(a)pyrene; Cell Line; Cell Transformation, Neoplastic; DNA; Epidermal Growth Factor; Metallothionein; Methylation; Methylnitronitrosoguanidine; Mice; Mice, Inbred BALB C; Mutation; Proto-Oncogenes; Tetradecanoylphorbol Acetate

The effects of all-trans retinoic acid (RA) on the growth and biochemical properties of five clonal strains of neoplastically transformed rat liver epithelial cells were studied. These cell strains were derived clonally from a single line of normal diploid rat liver epithelial cells that had been transformed by treatment with N-methyl-N'-nitro-N-nitrosoguanidine. The results show that RA induces inconsistent alterations in selected phenotypic properties of these five different cell strains. Retinoic acid either depressed, enhanced or produced no effect on the colony-forming ability in soft agar, on the activity of gamma-glutamyl transpeptidase, on the amount of cell-associated fibronectin, and on the binding capacity of 125I-epidermal growth factor (EGF). The only consistent correlation observed among cell strains was between the cellular ability to grow in soft agar and the amount of cell-associated fibronectin. Enhancement of anchorage-independent growth by retinoic acid was not mediated through changes in the number of EGF receptors. Our data demonstrate that the responses to retinoic acid of clonal subpopulations of chemically transformed rat liver epithelial cells are inconsistent, even when the clonal subpopulations are derived from a common precursor.

Topics: Animals; Cell Transformation, Neoplastic; Epithelium; ErbB Receptors; Fibronectins; gamma-Glutamyltransferase; Liver; Methylnitronitrosoguanidine; Rats; Tretinoin

A transfectable, presumably mutationally activated, c-Ha-ras gene was identified in a clonal population of 10T1/2 cells established from a Type II focus induced by exposure of a parental, wild-type population to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG).

Topics: Animals; Cell Line, Transformed; Cell Transformation, Neoplastic; Gene Expression Regulation; Genes; Genes, ras; Methylnitronitrosoguanidine; Mice; Mutation; Polymorphism, Restriction Fragment Length; Proto-Oncogene Proteins

Two-stage carcinogenesis is involved in the transformation of mouse fibroblasts BALB/c 3T3 cells. In order to investigate the role of cyclic adenosine 3':5'-monophosphate (cAMP)-dependent protein kinase at the stage of initiation, the following experiments were carried out: (a) two initiated clones (M14, M20) which exhibit 12-O-tetradecanoylphorbol-13-acetate-dependent growth in soft agar medium were isolated from cells treated with N-methyl-N'-nitro-N-nitrosoguanidine. The activity of cAMP-dependent protein kinase in M14 was reduced while that in M20 was similar to the level in parental cells. However, cAMP-binding activity to a regulatory subunit of cAMP-resistant clones were isolated from 4-nitroquinoline oxide- or ethyl methanesulfonate-treated cells. These clones have reduced activities in both cAMP-binding and cAMP-dependent protein kinase itself. Two of three cAMP-resistant clones were proved to be able to grow in soft agar medium only in the presence of 12-O-tetradecanoylphorbol-13-acetate.

Topics: 4-Nitroquinoline-1-oxide; Animals; Cell Division; Cell Line; Cell Transformation, Neoplastic; Clone Cells; Cyclic AMP; Ethyl Methanesulfonate; Fibroblasts; Methylnitronitrosoguanidine; Mice; Mice, Inbred BALB C; Protein Kinases; Tetradecanoylphorbol Acetate

The histology and ultrastructure of more than 100 tumors produced by a chemically transformed rat liver epithelial cell line and its single-cell-derived clonal subpopulations were studied. Wide-ranging morphologic presentations were observed, including carcinomas, sarcomas, "mixed epithelial-mesenchymal" tumors, and undifferentiated tumors. In addition to epidermoid and adenocarcinomas, several tumors were morphologically indistinguishable from hepatocellular carcinomas. The "mixed epithelial-mesenchymal" tumors reproduced most of the various histologic features of human hepatoblastomas. In many instances, the epithelial component occupied focal areas of tumors, and transmission electron microscopic studies of the "sarcomatous" regions revealed either spindle-shaped epithelial tumor cells or, in most cases, scattered epithelial tumor cells surrounded by numerous fibroblasts or myofibroblasts. Similar findings were observed in several "sarcomas" examined ultrastructurally, which suggests that most of the mixed tumors or sarcomas actually were spindle cell carcinomas and/or carcinomas with marked host fibroblastic reaction. However, in a few mixed tumors produced by clonally derived cell strains, unequivocal carcinosarcomas with neoplastic osteoid or chondroid tissue were demonstrated. The findings of this study are discussed in the context of current insights on the cellular composition of the liver and on the histogenesis of human hepatoblastoma.

Topics: Adenoma, Bile Duct; Animals; Cell Line; Cell Transformation, Neoplastic; Liver Neoplasms; Liver Neoplasms, Experimental; Methylnitronitrosoguanidine; Microscopy, Electron; Neoplasm Metastasis; Neoplasm Transplantation; Rats; Sarcoma, Experimental

The cell kinetic alteration in the background mucosa of canine gastric cancer induced by N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) was evaluated by cytofluorometry in which the rate of S and G2 + M phase cell in gastric mucosal cells could be calculated, and a triphasic alteration was demonstrated; an initial reduction phase, an increase phase and a plateau phase with a high value. The initial reduction phase was caused by non-specific toxicity of ENNG as observed in drug induced gastric mucosal lesions, and subsequent increase and plateau phases originated from the action of ENNG itself to activate the mucosal turn-over and from histological changes in the background mucosa such as regenerative hyperplastic change after mucosal erosion and atrophic changes, sometimes including intestinal metaplastic change. Further, in comparison to carcinogenesis in chemically induced gastric cancer with and without a surfactant (Tween 60), it was suggested that one of the promotion effects of Tween 60 was closely related with activation of the mucosal turn-over.

Topics: Adenocarcinoma, Mucinous; Animals; Cell Cycle; Cell Transformation, Neoplastic; DNA, Neoplasm; Dogs; Flow Cytometry; Gastric Mucosa; Gastroscopy; Male; Methylnitronitrosoguanidine; Polysorbates; RNA, Neoplasm; Stomach Neoplasms

Unscheduled DNA synthesis (UDS) was studied by quantitative autoradiography in human urothelial cells of three transformation grades (TGr I-III). Cells incubated in arginine-free medium supplemented with hydroxyurea showed dose-dependent UDS after administration of agents injurious to DNA, while the scheduled synthesis of DNA was nearly totally suppressed. UDS was demonstrated after treatment with the ultimate carcinogen N-methyl-N-nitroso-N'-nitroguanidine (CAS: 70-25-7) or with the procarcinogen 4-nitroquinoline-1-oxide (4-NQO; CAS: 56-57-5). In cultures treated with benzo[a]pyrene (CAS: 50-32-8), which requires a different activation system than that for 4-NQO, UDS was less pronounced. In 9 cell lines the average rates of UDS were inversely related to TGr. Two cell lines showed a different pattern.

Topics: 4-Nitroquinoline-1-oxide; Autoradiography; Carcinogens; Cell Line; Cell Transformation, Neoplastic; DNA Repair; Dose-Response Relationship, Drug; Epithelial Cells; Humans; Methylnitronitrosoguanidine; Urinary Bladder

In mutation and cell transformation assays, it has long been recognized that the common practice of using different numbers of cells on dishes with or without selective conditions creates a source of bias in mutant fraction determination. This is simply because colony formation may be enhanced or suppressed at higher initial cell densities, depending on the assay and agent tested. We propose a solution that consists of the inclusion of an experimentally distinguishable population of cells as an internal standard for colony-forming ability at the high cell density required for detection of rare variants. This method is found to be highly satisfactory for use in measuring mutation to 6-thioguanine resistance in a diploid human B lymphoblast line. For treatment with anti-2,3-dihydroxy-1,10b-epoxy-1,2,3-trihydrofluoranthene (FDE), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and 4-nitroquinoline-oxide (4NQO), the calculated induced mutant fractions using the internal-standard method were significantly lower than those calculated using the conventional low-density-plating efficiency method. The results of these experiments and our analysis lead us to conclude that this approach is applicable to all single cell mutation or transformation assays and is a necessary feature of assays in which an accurate knowledge of the fraction of rare variants is required.

Topics: 4-Nitroquinoline-1-oxide; Adenine Phosphoribosyltransferase; B-Lymphocytes; Cell Line; Cell Survival; Cell Transformation, Neoplastic; Child, Preschool; Colony-Forming Units Assay; Drug Resistance; Fluorenes; Humans; Hypoxanthine Phosphoribosyltransferase; Male; Methylnitronitrosoguanidine; Mutagenicity Tests; Thioguanine

A method has been developed by which to amplify expression of phenotypic transformation of C3H/10T1/2 clone 8 mouse embryo cells not otherwise observed in the standard transformation assay. The expression of transformed foci was amplified by subcultivating chemically treated target cells after they had reached confluence and replating them at subconfluent cell densities. Conditions leading to the expression of the highest numbers of transformed foci include a) a cell seeding density for chemical treatment of 1 X 10(4) cells/dish, b) subculture 4 weeks after treatment, and c) replating cells at a density of 2 X 10(5) cells/-dish. Agents capable of inducing transformation in the standard assay (e.g., 4,4'-bis(dimethylamino)benzophenone, benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene, and others) also yielded transformation in the replating assay. The more marginal transforming activities of chemicals such as ethyl methanesulfonate, 7-(bromomethyl)-12-methylbenz[a]anthracene, and N-methyl-N'-nitro-N-nitrosoguanidine were enhanced by the amplification procedure. Compounds that failed to elicit focal transformation in the standard assay (e.g., dibenz[a,h]anthracene, Tris(2,3-dibromopropyl) phosphate, lead acetate, benzidine, propyleneimine, N-hydroxy-2-fluorenylacetamide, and numerous other compounds of various chemical classes) induced significant levels of phenotypic transformation upon amplification. Noncarcinogens (e.g., phenanthrene, anthracene, 2-aminobiphenyl, cycloheximide, and others) failed to cause significant phenotypic transformation even when cells were replated. To further enhance the applicability of this new replating system, an exogenous source of metabolic activation was added: a 9,000 X g supernatant from Aroclor 1254-induced rat hepatic S-9. This activation system was found a) to be only minimally cytotoxic by itself and b) to be able to mediate NADPH-dependent, dose-dependent toxicity, and transformation by activating the procarcinogens dimethylnitrosamine, 2-naphthylamine, 2-aminoanthracene, and aflatoxin B1. With the use of this revised assay, 14 coded and 23 model compounds were tested. Agreement with in vivo results was observed to be over 85%. The marked sensitivity and discriminatory ability of this revised assay procedure suggest its usefulness as a screen for potential carcinogens of diverse chemical structure.

Topics: 2-Naphthylamine; Aflatoxin B1; Aflatoxins; Animals; Biotransformation; Carcinogens; Cell Transformation, Neoplastic; Clone Cells; Diethylnitrosamine; Dimethylnitrosamine; Male; Methylcholanthrene; Methylnitronitrosoguanidine; Rats; Rats, Inbred F344

Two murine monoclonal antibodies, 3BG8 and 9BG8, which were raised against a rat tracheal squamous-cell-carcinoma cell line, recognize cell-surface antigens on normal rat squamous epithelium (skin, esophagus, vagina, and cornea) as well as on carcinogen-exposed, immortalized, rat tracheal epithelial cells. Monoclonal antibody 3BG8 binds to a 115-kilodalton cell-surface protein on undifferentiated basal cells of the epithelium, while the binding of the other antibody, 9BG8, occurs in both differentiated and undifferentiated populations of normal squamous epithelium and squamous cell carcinomas. Undifferentiated tracheal carcinomas bound only the 3BG8 antibody. No binding of either antibody was detected on normal tracheal mucociliary epithelium. Only under conditions that induce squamous differentiation of rat tracheal epithelium was binding of 3BG8 and 9BG8 detected. For reasons which are not clear at present, 9BG8 dramatically inhibits the growth of normal tracheal and esophageal cells in primary culture, whereas only 3BG8 affects the growth of carcinogen-altered tracheal cell lines. Based on antigen characterization and distribution, it is concluded that the 3BG8 and 9BG8 epitopes are localized on differentiation antigens which differ from others that have been previously described.

Topics: Animals; Antibodies, Monoclonal; Antigen-Antibody Complex; Antigens, Neoplasm; Antigens, Surface; Cell Line; Cell Transformation, Neoplastic; Cells, Cultured; Epithelial Cells; Epithelium; Fluorescent Antibody Technique; Methylnitronitrosoguanidine; Rats

Topics: Animals; Cell Count; Cell Transformation, Neoplastic; Culture Media; Fibroblasts; Methylnitronitrosoguanidine; Rats

Following carcinogen exposure in vitro, normal rat tracheal epithelial cells are transformed in a multistage process in which the cultured cells become immortal and, ultimately, neoplastic. Five cell lines derived from tumors produced by neoplastically transformed rat tracheal epithelial cells were examined for the expression of 11 cellular oncogenes previously implicated in pulmonary or epithelial carcinogenesis. RNA homologous to fms was expressed at a level 5-19 times higher than normal tracheal epithelial cells in three of five of the tumor-derived lines. All three lines expressing high levels of fms-related RNA gave rise to invasive tumors of epithelial origin when injected into nude mice. Increased expression of the fms-related mRNA was not due to gene amplification, and no gene rearrangement was detected by Southern analyses. RNA blot analysis using a 3' v-fms probe detected a 9.5-kilobase message in the three tumor-derived lines, whereas both normal rat alveolar macrophages and the human choriocarcinoma line BeWo expressed a fms transcript of approximately 4 kilobases. We conclude from these data that the gene expressed as a 9.5-kilobase transcript in these neoplastic epithelial cells is a member of a fms-related gene family but may be distinct from the gene that encodes the macrophage colony-stimulating factor (CSF-1) receptor.

Topics: Animals; Base Sequence; Carcinoma, Squamous Cell; Cell Line; Cell Transformation, Neoplastic; Epithelium; Gamma Rays; Gene Amplification; Genes; Methylnitronitrosoguanidine; Oncogenes; Protein-Tyrosine Kinases; Rats; Trachea; Tracheal Neoplasms

We studied whether arsenic, nickel, and chromium compounds that are human carcinogens could induce transformation of cultured primary human diploid foreskin cells (HFC). All nickel compounds tested, PbCrO4, K2Cr2O7, CrO3, Na2HAsO4, NaAsO2, and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) caused significant (p = 0.001) dose-dependent inductions of anchorage-independent colonies in HFC. KH2AsO4, CaCl2, MnCl2, and Hg(CH3CO2)2 did not induce anchorage independence. Optimal expression times for induction of anchorage independence in HFC were observed as early as 11 days following treatment with MNNG, Ni3S2, Ni(C2H3O2), or NiSO4. Cell strains derived from anchorage-independent colonies showed 33 to 429-fold higher plating efficiencies in soft agar than parental populations, and the anchorage-independent phenotype was stable for eight passages, at which time cells senesced. Anchorage-independent cell strains derived from metal salt-treated cells were not resistant to the cytotoxicity of metal salts, indicating metal salts induced rather than selected for anchorage independence. Nine of 10 cell strains derived from metal compound- or MNNG-induced anchorage-independent colonies displayed the same or lower saturation densities than untreated human fibroblasts. None of these cell strains escaped senescence or showed definitive morphological transformation. MNNG (1 micrograms/ml) induced anchorage independence and mutation to ouabain resistance and 6-thioguanine resistance in HFC, but concentrations of Ni2S3 that induced anchorage independence did not induce mutation at either locus in HFC. These results demonstrate that carcinogenic metal salts induce stable anchorage independence early in human diploid foreskin fibroblasts, and this anchorage independence is independent of other in vitro markers of fibroblast transformation, such as focus formation or immortality. Metal salt induction of anchorage independence can now be used as an assay to study mechanisms of genotoxicity exerted by carcinogenic metal compounds in human cells.

Topics: Acetates; Acetic Acid; Arsenates; Calcium Chloride; Carcinogens; Cell Adhesion; Cell Line; Cell Transformation, Neoplastic; Chlorides; Chromates; Fibroblasts; Guanosine; Humans; Manganese; Manganese Compounds; Mercury; Metals; Methylnitronitrosoguanidine; Mutation; Nickel; Skin; Thionucleosides

N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was given to 3 groups of rats with the drinking water for 32 weeks in different doses: 25, 50 and 100 micrograms/ml. After 50 weeks the induced tumours of the stomach and the upper small intestine were investigated. Most tumours were well differentiated adenocarcinomas or adenomatous-hyperplastic lesions with focal adenocarcinoma. After low MNNG-concentration (25 micrograms/ml) only adenomatous hyperplastic lesions with focal adenocarcinoma were found. A tumour development in connection with intestinal metaplasia was detectable exclusively in two rats of the group receiving 50 micrograms MNNG/ml. The frequency of gastric tumours shows a relatively low peak (3.3 tumours/10 animals) after administering a medium MNNG-concentration (50 micrograms/ml) and a little decrease of the frequency after higher MNNG-concentration, as opposed to the approximately linear dose-related increase of the tumour frequency in the upper small intestine. The highest tumour induction rate was found in the upper small intestine after 100 micrograms MNNG/ml (5.6 tumours/10 rats). It can be concluded that the mucosa of the upper small intestine possesses a greater susceptibility to the carcinogenic effect of MNNG than the glandular stomach of the rat.

Topics: Adenocarcinoma; Administration, Oral; Animals; Cell Transformation, Neoplastic; Dose-Response Relationship, Drug; Hyperplasia; Intestinal Neoplasms; Intestine, Small; Male; Methylnitronitrosoguanidine; Neoplasm Invasiveness; Rats; Rats, Inbred Strains; Stomach; Stomach Neoplasms

Few of the seventy-five chlorinated dibenzo-p-dioxin isomers present in the environment have been adequately characterized for their carcinogenic potential. In previous studies we observed that the carcinogenic dioxin isomer 2,3,7,8-tetrachlorodibenzo-p-dioxin promoted cell transformation when continuously applied to C3H/10T1/2 cells initiated by treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The current study was undertaken to evaluate the response of the C3H/10T1/2 cell transformation system to several other dioxin isomers of known carcinogenic potential. 1,2,3,6,7,8- and 1,2,3,7,8,9-hexachlorodibenzo-p-dioxin (HCDD) (1-1000 nM) and 2,7-dichlorodibenzo-p-dioxin (0.1-20 microM) failed to transform C3H/10T1/2 cells or to initiate transformation in cultures subsequently treated with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate. Continuous exposure of MNNG-initiated cultures to 2,7-DCDD (1-5000 nM) produced elevated but not statistically significant numbers of transformed foci at the highest dose tested. 1,2,3,6,7,8- and 1,2,3,7,8,9-HCDD were promoters of transformation when applied at concentrations greater than or equal to 12 and 40 pM, respectively, to C3H/10T1/2 cultures initiated with MNNG. Maximum responses for both HCDD isomers were attained at concentrations between 120 and 400 pM. These studies suggest that the C3H/10T1/2 cell transformation system may provide a relevant in vitro model for the identification and study of carcinogenic dioxin isomers.

Topics: Animals; Cell Transformation, Neoplastic; Dioxins; Dose-Response Relationship, Drug; Isomerism; Methylnitronitrosoguanidine; Mice; Mice, Inbred C3H; Mutagenicity Tests; Polychlorinated Dibenzodioxins

Topics: Cell Transformation, Neoplastic; Cells, Cultured; Fetus; Fibroblasts; Humans; Lung; Methylnitronitrosoguanidine

We determined the nucleotide sequence of the rearranged trp-met genomic locus and the corresponding portions of the unrearranged tpr and met genomic fragments. The breakpoints occur at one end of a stretch of 21 A residues that follow an Alu repetitive sequence in the tpr locus and within a group of 3 A residues in the met proto-oncogene locus. We conclude that the fusion between the tpr locus on chromosome 1 and the met locus on chromosome 7 resulted from a recombination event.

Topics: Base Sequence; Cell Transformation, Neoplastic; Chromosomes, Human, Pair 1; Chromosomes, Human, Pair 7; DNA, Neoplasm; Gene Expression Regulation; Humans; Methylnitronitrosoguanidine; Proto-Oncogene Mas; Proto-Oncogenes; Recombination, Genetic; Repetitive Sequences, Nucleic Acid; Translocation, Genetic

Efforts to investigate the progression of events that lead human cells of epithelial origin to become neoplastic in response to carcinogenic agents have been aided by the development of tissue culture systems for propagation of epithelial cells. In the present study, nontumorigenic human epidermal keratinocytes immortalized by adenovirus 12 and simian virus 40 (Ad 12-SV40) were transformed by treatment with the chemical carcinogens N-methyl-N'-nitro-N-nitrosoguanidine or 4-nitroquinoline-1-oxide. Such transformants showed morphological alterations and induced carcinomas when transplanted into nude mice, whereas primary human epidermal keratinocytes treated with these chemical carcinogens failed to show any evidence of transformation. This in vitro system may be useful in assessing environmental carcinogens for human epithelial cells and in detecting new human oncogenes.

Topics: 4-Nitroquinoline-1-oxide; Adenoviruses, Human; Animals; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Epidermal Cells; Humans; Keratins; Methylnitronitrosoguanidine; Mice; Mice, Nude; Neoplasm Transplantation; Nitroquinolines; Oncogenes; Simian virus 40; Skin Neoplasms

The ability of the hypomethylating agent 5-azacytidine to transform normal primary rat tracheal epithelial cells was determined. A single 24-h exposure to 5-azacytidine resulted in the transformation of rat tracheal epithelial cells at concentrations of the cytosine analogue ranging from 1 to 4 microM. Doses of 5-azacytidine that produced these enhanced growth transformants were nonmutagenic as measured by the induction of 6-thioguanine or ouabain resistance. In addition, the hypomethylating agent 5-azadeoxycytidine also transformed primary rat tracheal epithelial cells, whereas 6-azacytidine, a cytosine analogue which does not induce DNA hypomethylation, did not. Enhanced growth transformants resulting from 5-azacytidine exposure continued to progress in the absence of further treatment to give rise to variants with indefinite growth capacity which ultimately became neoplastic. These results indicate that hypomethylating agents can transform normal cells and suggest that changes in DNA methylation may occur during the initial stages of neoplastic transformation.

Topics: Animals; Azacitidine; Cell Transformation, Neoplastic; Cells, Cultured; Decitabine; DNA; Dose-Response Relationship, Drug; Epithelium; Male; Methylation; Methylnitronitrosoguanidine; Mutation; Rats; Rats, Inbred F344; Time Factors; Trachea

Pepsinogen mRNA is shown to be the major fraction of rat poly (A)+RNA. It codes polypeptide with molecular weight of about 45 kD. Changes in the pepsinogen mRNA content at the early stage of carcinogenesis are nonspecific and are due to the toxic effect of MNNG. Steady shifts in the quantity of pepsinogen mRNA are found between the 1st and 3d months. Pepsinogen mRNA content decreases down to the half of the normal one between the 3d and 6th months. A quantity of RNA capable to be a template for pepsinogen synthesis is reduced by more than 90% in the MNNG induced tumour. The pepsinogen production defect in gastric mucosa neoplasia is mainly due to pepsinogen mRNA synthesis damage.

Topics: Animals; Cell Transformation, Neoplastic; Gastric Mucosa; Methylnitronitrosoguanidine; Pepsinogens; Poly A; Protein Biosynthesis; Rats; RNA; RNA, Messenger; Stomach Neoplasms; Time Factors

Topics: Adult; Cell Transformation, Neoplastic; Cells, Cultured; Esophageal Neoplasms; Fibroblasts; Humans; Methylnitronitrosoguanidine; Middle Aged

The early passage diploid Syrian hamster embryo (SHE) cells were treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The treated cells proliferated rapidly; the doubling times shortened; colonies appeared in solid agar medium and transformed foci formed in tissue culture. All of these phenomena suggest that malignant transformation of SHE cell has occurred. Faster cell division rate and multipolar mitosis were demonstrated by time-lapse cinemicrography and scanning electron microscopy. Multipolar mitosis can occur in two forms: direct division and indirect division. The transformed cells were more abundant in microvilli, the number of which increased in accordance with the degree of malignancy. In comparison with the controls, the transformed cells expressed a greater tritiated thymidine incorporation, greater DNA contents and more chromosomes, but no difference in nuclear area. The determination of amino acid changes in media due to the growth of transformed cells showed that the decrease in arginine and increase in ornithine are significant. The results of allogenic animal inoculation suggest that the transformed cells can be characterized into several different stages in the process of transformation.

Topics: Animals; Cell Division; Cell Line; Cell Transformation, Neoplastic; Chromosome Aberrations; Concanavalin A; Cricetinae; Cyclic AMP; DNA, Neoplasm; Embryo, Mammalian; Mesocricetus; Methylnitronitrosoguanidine; Microscopy, Electron, Scanning; Neoplasm Transplantation

When cells were exposed simultaneously for a 24-h period to the poly(ADP-ribose) synthetase inhibitor 3-aminobenzamide (3AB) (1 or 3 mM) plus aflatoxin B1 (AfB1), no increase in toxicity and limited enhancement of transformation frequency (less than 2 X) was observed. Similarly, simultaneous treatment of cell with 3AB plus methylcholanthrene (MCA) had limited effects, slightly decreasing both toxicity and transformation. In contrast, simultaneous treatment with non-toxic doses of 3AB together with the alkylating agents N-methyl-N'-N-nitro-nitrosoguanidine (MNNG) or ethyl methanesulfonate (EMS) resulted in substantial enhancement of the toxicity and transforming effects of both short-chain alkylating agents. When EMS and varying doses of 3AB (0.1-3 mM) were administered simultaneously for 24 h, increasing levels of 3AB were found to cause a dose-dependent enhancement in toxicity and transformation. To explore the relationship of MNNG- and 3AB-induced effects, two further experiments were performed. First, cells were treated with MNNG plus 3AB for varying lengths of time (4, 24, 72 h). Although exposure for as little as 4 h enhanced toxicity and transformation, these effects were even more profound following 24 or 72 h exposure. Second, cells were exposed to 3AB for varing times prior to or after MNNG exposure. Under these conditions the addition of 3AB up to 6 h post MNNG exposure caused profound enhancement of toxicity and transformation, whereas addition of 3AB 24 h post exposure had minimal effects. Thus the co-carcinogenic effect of 3AB is agent-specific, time-specific and dose-dependent.

Topics: Aflatoxin B1; Aflatoxins; Animals; Benzamides; Carcinogens; Cell Transformation, Neoplastic; Cells, Cultured; Clone Cells; Ethyl Methanesulfonate; Kinetics; Methylcholanthrene; Methylnitronitrosoguanidine; Mice; Mice, Inbred BALB C

Inhibition of poly(ADP-Rib) by benzamide (BA) or 3-amino-benzamide (3AB) for a limited period (i.e., when ADP-ribosylation is elevated) during and shortly following X-ray or MNNG-induced DNA damage of BALB/3T3 cells significantly (3- to 30-fold) enhanced transformation frequency by these agents. Individual Type III foci isolated from benzamide, X-ray, or X-ray plus benzamide treated cultures were established and characterized for growth in soft agar and for tumor induction in nude mice. DNA isolated from representative transformed lines established as a result of BA, X-ray or X-ray and BA treatments was transfected onto NIH/3T3 cells. Transformation efficiencies ranging from 0.17 to 0.28 foci/micrograms of DNA were observed suggesting the possibility that dominant transforming gene(s) were responsible for the oncogenic phenotype of radiation and benzamide transformed DNA.

Topics: Animals; Base Sequence; Benzamides; Cell Survival; Cell Transformation, Neoplastic; Cells, Cultured; DNA; DNA, Neoplasm; Methylnitronitrosoguanidine; Mice; NAD; Oncogenes; Poly(ADP-ribose) Polymerase Inhibitors

Oncogenic transformation of mouse 10T 1/2 fibroblasts induced upon exposure to X-ray or N-methyl-N'-nitro-N-nitrosoguanidine was suppressed if lipopolysaccharide (LPS) was present in the culture medium. The suppressive effect of LPS was exerted within 24 h after irradiation. Suppression was dependent on the concentration of LPS added and LPS (2 micrograms/ml) derived from Salmonella minnesota R595 reduced the number of transformed type III foci per dish from 0.39 to 0.15. Indomethacin (1 to 30 microM) further enhanced the effect of LPS in a dose-dependent manner.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Cell Transformation, Neoplastic; Cells, Cultured; Fibroblasts; Indomethacin; Lipopolysaccharides; Methylnitronitrosoguanidine; Mice; Mice, Inbred C3H; Neoplasms, Experimental; Neoplasms, Radiation-Induced; X-Rays

The intralaboratory and interlaboratory reproducibility of a DNA virus (SA7) transformation enhancement assay was investigated using nine carcinogenic and noncarcinogenic compounds representing a variety of chemical classes. By the use of standardized procedures designed to limit assay variables, replicate assay data were collected in two independent laboratories and analyzed for concurrence. The carcinogens, 7,12-dimethylbenz(a)anthracene, benzo(a)pyrene, and N-methyl-N'-nitro-N-nitrosoguanidine yielded reproducible dose-dependent cytotoxicity and positive transformation effects (defined as statistically significant [p less than or equal to 0.05] enhancement of virus transformation at two or more consecutive dose levels) in all experiments in both laboratories. The carcinogens lead chromate, diethylnitrosamine, 4-nitroquinoline-N-oxide, and 2-acetylaminofluorene demonstrated enhancement of SA7 transformation at two or more dose levels in 40-50% of the assays. The noncarcinogenic structural analogs anthracene and pyrene consistently did not produce positive assay responses when tested at dose levels up to the limits of solubility. Good interlaboratory concurrence was demonstrated for these model compounds in the Syrian hamster embryo cell-SA7 assay.

Topics: 2-Acetylaminofluorene; 4-Nitroquinoline-1-oxide; 9,10-Dimethyl-1,2-benzanthracene; Adenoviridae; Adenoviruses, Simian; Animals; Benzo(a)pyrene; Carcinogens; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cells, Cultured; Chromates; Cocarcinogenesis; Cricetinae; Diethylnitrosamine; Dose-Response Relationship, Drug; Embryo, Mammalian; Lead; Mesocricetus; Methylnitronitrosoguanidine

We have investigated whether the activation of endogenous ras genes is associated with the immortalization or malignant transformation of primary hamster epidermal cells by chemical carcinogens. We have also asked whether transfection of a cloned c-Ha-ras oncogene (pEJ) into a nontumorigenic cell line established from hamster epidermal cells by N-methyl-N'-nitro-N-nitrosoguanidine treatment can induce conversion to a malignant phenotype. DNA from the nontumorigenic epidermal cell line (H5-MNNG) and from two neoplastic cell lines transformed by benzo(a)pyrene was not capable of transforming NIH/3T3 cells. This result suggests that these cells do not contain an activated (mutated) ras gene. However, when H5-MNNG cells were cotransfected with pEJ and pSV2-gpt, a plasmid containing the dominant selectable marker gene Ecogpt, seven of nine clones of Ecogpt transformants formed carcinomas in nude mice and colonies in soft agar. Southern blot analysis of BamHl-digested genomic DNA from the Ecogpt-transformed clones indicated that rapid malignant transformation was associated with integration of a complete copy of the 6.6-kilobase fragment of pEJ containing the activated c-Ha-ras gene. Furthermore, DNA from the malignant clones transformed NIH/3T3 cells in a secondary transfection assay. These studies demonstrate that a mutated c-Ha-ras gene, under the transcriptional control of its normal cellular promoter, can rapidly transform a nontumorigenic epidermal cell line. This result suggests that activation of an endogenous c-ras gene can function as the final completing event in the progression of epithelial cells to the malignant phenotype. Thus, preneoplastic cell lines of both mesenchymal and epithelial origin have now been shown to be susceptible to malignant conversion by a single mutation in a c-Ha-ras proto-oncogene.

Topics: Animals; Carcinogens; Cell Line; Cell Transformation, Neoplastic; Cricetinae; Epidermis; Gene Expression Regulation; Humans; Methylnitronitrosoguanidine; Mutation; Oncogenes; Proto-Oncogene Mas; Proto-Oncogene Proteins; Transfection; Urinary Bladder Neoplasms

An orthogonal design method was used to study two-stage chemical carcinogenesis in BALB/3T3 cells. Four factors were studied: different concentrations of the initiator N-methyl-N-nitro-N'-nitrosoguanidine (MNNG); different concentrations of the promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA); different concentrations of fetal calf serum (FCS) and different times at which TPA was added after cell initiation. From the results of three experiments designed by Table L9(3(4)), L8(2(7)) and L16(4(5)), 0.3 microgram/ml MNNG was the highest possible initiating concentration and 0.25 microgram/ml TPA was the minimum effective concentration for promoting activity. There is synergy between MNNG and TPA, the optimum combination in sequential treatment being 0.3 microgram/ml MNNG and 0.25 microgram/ml TPA. The 10% FCS standard concentration was the optimal one; however, below 5% few foci appeared. The time at which TPA was added had little effect on cloning efficiency and transformation frequency. So the use of this orthogonal design in cell culture has many advantages: several factors can be tested simultaneously; it is easy to find the optimal protocol conditions and the dose-response relationship is stable, which enables the reproducibility to be improved. In addition, the different tables proposed may help to reveal unexpected problems.

Topics: Animals; Cell Transformation, Neoplastic; Cells, Cultured; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Fetal Blood; Methylnitronitrosoguanidine; Mice; Statistics as Topic; Tetradecanoylphorbol Acetate

The purpose of the studies described in this and the accompanying paper (D.J. Fitzgerald et al., Cancer Res., 46:4642-4649, 1986) was to define the transformed colonies of the rat tracheal epithelial cell transformation system (see P. Nettesheim and J.C. Barrett, CRC Crit. Rev. Toxicol., 12: 215-239, 1984) in terms of differentiation and growth characteristics. Exposure of low-density primary rat tracheal epithelial cell cultures to N-methyl-N'-nitro-N-nitrosoguanidine results in the development of growth-altered colonies of different sizes and morphologies, which can be readily scored 5 wk after treatment. These colonies were classified into four morphological types (I to IV) based on their light microscopic and ultrastructural features. Colonies designated as types I and II were small, had a low cell density, and were composed principally of large, pale-staining (Giemsa) flattened cells with a low nuclear/cytoplasmic ratio. Examination of the fine structure of these colonies revealed a monolayer of extremely attenuated cells containing well-developed Golgi complexes, endoplasmic reticulum, numerous secondary lysosomes, but few tonofilaments and desmosomes. Colonies of types III and IV were large, basophilic (Giemsa), high-density colonies, consisting primarily of closely packed, small, round cells with high nuclear/cytoplasmic ratio. Analysis of the fine structure of these colonies revealed two to four stratified layers of poorly differentiated cells and cells having features of keratinocyte differentiation, as evidence by abundant tonofilament bundles, well-developed desmosomes, and occasional keratohyaline granules. Differential cell counts were carried out at the ultrastructural level on pooled cells from each colony type (except type I); approximately 90% of the cells from type II colonies were large, electron-lucent cells, while this cell type comprised only 20 to 30% of the cells of colonies of types III and IV. The predominant cell type (approximately 60%) in colonies of types III and IV showed clear signs of keratinocyte differentiation. Type IV colonies contained a significantly larger proportion (greater than 20%) of small, poorly differentiated cells than type III (6%) and type II (1%) colonies. A combined autoradiographic and ultrastructural study revealed that the small, poorly differentiated cells were most active in synthesizing DNA and had a labeling index of 60%. Other cell types were far less frequently labeled, particularly the large,

Topics: Animals; Cell Cycle; Cell Transformation, Neoplastic; Cells, Cultured; Epithelium; Methylnitronitrosoguanidine; Microscopy, Electron; Microscopy, Electron, Scanning; Rats; Time Factors; Tracheal Neoplasms

The purpose of the studies described here was to define the biological behavior of the various clonally transformed colonies observed in cultures of carcinogen-exposed rat tracheal epithelial cells. As described in the preceding paper (H. Kitamura et al., Cancer Res., 46: 4631-4641, 1986), these colonies fall into four morphologically distinct categories. In the studies reported here we found that type I colonies had the smallest growth fraction (7%) and contained the lowest frequency of clonogenic cells (approximately 10(-3)). Colonies of types II to IV had mean growth fractions of 21 to 28%, and the frequency of clonogenic cells was 2 to 5 X 10(-2) when measured under growth-permissive conditions (3T3 feeders). When the clonogenic cell assays were performed under selective conditions to identify cell variants which can grow without feeder support, the average frequency of such clonogenic cells in type I colonies was less than 4 X 10(-5) and in colonies of types II to IV, between 5 X 10(-4) and 10(-2). In type IV colonies, the total number of cells per colony increased 8-fold between 5 and 12 wk postcarcinogen, but the clonogenic cell compartment increased 42-fold; the compartment of variant clonogenic cells, which are able to replicate on plastic, increased 139-fold during the same period of time. This indicated that major changes in the self-renewal capacity of the clonogenic cells were taking place during this early stage of transformation. Examination of the daughter colonies produced by replating colonies of types I to IV revealed that clonogenic cells with different growth potential existed within the same parent colony. Comparison of transformed colonies of the same type showed a marked degree of heterogeneity in the sizes of growth fractions and clonogenic cell fractions. These studies further indicated that, within all colonies, including the most advanced transformants, the majority of the cells were nonreplicating, terminal cells, suggesting that, at least during early stages of transformation, the transformed characteristics were not transferred from parent to daughter cells. With the exception of type I colonies, most of the colonies recognizable at 5 wk after carcinogen exposure progressed with time and acquired the morphological characteristics of type IV colonies, which were the most transformed phenotype. We conclude that transformation of rat tracheal epithelial cells is an asynchronous process and that the morphologically distinct type

Topics: Animals; Cell Cycle; Cell Transformation, Neoplastic; Epithelium; Methylnitronitrosoguanidine; Neoplastic Stem Cells; Rats; Time Factors; Tracheal Neoplasms

Retinoic acid (RA) treatment of rat tracheal epithelial (RTE) cells, pre-exposed to the direct-acting carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), inhibited transformation in a dose-dependent manner. Treatment with RA at concentrations ranging from 3-33 nM reduced the MNNG-induced transformation frequencies by 13-81% and in some experiments by greater than 90%. RA treatment for only 3 days caused 65-75% inhibition of transformation; treatments of longer duration resulted in greater inhibition of transformation. Delaying the onset of RA treatment reduced its effectiveness, but even when RA treatment was delayed for 3 weeks following MNNG exposure, 60% inhibition still occurred. The inhibition of transformation appeared to be irreversible. The colony forming efficiency of cells isolated from transformed colonies 5 weeks after MNNG exposure was drastically reduced when the replated cells were treated with RA either 1 day or 4 days after plating, indicating that RA blocked cell replication. However, cells isolated from transformed colonies at later times after MNNG exposure were increasingly resistant to the antiproliferative effects of RA. The RA concentration causing 50% inhibition (RA-IC50) of colony formation was 0.1-0.3 nM for cells isolated from 3-5 week-old transformed colonies; it increased greater than 100-fold for cells isolated from 12-week-old transformants. Five established RTE cell lines also showed a much increased resistance to the antiproliferative effects of RA; two of these cell lines were even slightly stimulated in their colony forming ability by RA. The RA-IC50 of colony forming efficiency of normal RTE cells was also determined and compared to that of cells isolated from 5-week-old transformed colonies. Since the normal RTE cells require 3T3 feeder layers for clonal growth, both cell types were grown on feeders. For both cell types, the RA-IC50 was similar (150-300 nM); the requirement for relatively high RA concentrations was attributed in part to the rapid RA metabolism by the feeder cells. These experiments show that early RTE cell transformants are growth-inhibited by RA; however, they increasingly lose their sensitivity to the growth controlling effects of RA as they progress to a more advanced stage of transformation. The inhibition of tracheal cell transformation by RA is probably due to the antiproliferative effects of the retinoid.

Topics: Animals; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Epithelium; Male; Methylnitronitrosoguanidine; Rats; Rats, Inbred F344; Trachea; Tretinoin

Treatment of C3H/10T1/2 mouse embryo fibroblasts with N-methyl-N'-nitro-N-nitrosoguanidine and then 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in the production of numerous foci of morphologically transformed cells. When dishes containing foci were provided medium which did not contain TPA, up to 84% of the foci were found to regress. Promotion of morphological transformation by TPA in C3H/10T1/2 cells may thus be a reversible process.

Topics: Animals; Cell Transformation, Neoplastic; Cells, Cultured; Methylnitronitrosoguanidine; Mice; Phenotype; Tetradecanoylphorbol Acetate

A serum- and feeder cell-free medium has been developed for the proliferation of rat tracheal epithelial (RTE) cells at clonal density. In this medium, RTE cells continue to proliferate for several weeks after cells in serum containing medium on feeder cells have begun to differentiate. The responsiveness of RTE cells to selected hormones and growth factors was determined using a clonal growth assay. The colony-forming efficiency (CFE) of RTE cells was reduced greater than 85% when bovine pituitary extract or bovine serum albumin were deleted from the medium and 45-70% reductions in CFE were observed when insulin, hydrocortisone, epidermal growth factor or cholera toxin were deleted. RTE cells also require high concentrations of Ca2+ (0.8 mM) for maximal clonal proliferation in this medium. The induction by carcinogens of preneoplastic RTE cell variants resistant to serum-mediated squamous differentiation was compared in serum-free medium and in serum-containing medium on feeder cells. N-methyl-N'-nitro-N-nitrosoguanidine was considerably more cytotoxic and effective as a transforming agent on an equivalent dose basis for RTE cells in serum-free medium. In contrast, (+/-)-7B,8-dihydroxy-9,10-epoxy-7,8,9, 10-tetrahydrobenzo[a]pyrene was equally cytotoxic and transforming under both culture conditions. This serum-free culture system for primary RTE cells will be useful in studies on the control of normal epithelial cell proliferation and differentiation by defined growth factors and in studies on the cellular changes involved in carcinogenesis.

Topics: Animals; Blood Physiological Phenomena; Calcium; Cell Differentiation; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Culture Media; Epithelial Cells; Epithelium; Growth Substances; Hormones; Male; Methylnitronitrosoguanidine; Rats; Rats, Inbred F344; Trachea

We have raised antibodies against a synthetic dodecapeptide corresponding to the carboxyl terminus of the predicted met gene product. Phosphorylation of 60 kDa and 65 kDa proteins on tyrosine residues was observed when immunoprecipitates of cells containing the activated human met gene were incubated with [gamma-32P]ATP. Phosphoproteins with the same molecular masses could be immunoprecipitated from cells metabolically labelled with [32P]orthophosphate. When considered together, these observations indicate that the activated human met gene encodes 60 kDa and 65 kDa proteins that can catalyse autophosphorylation on tyrosine residues.

Topics: Amino Acid Sequence; Animals; Antibodies; Cell Line; Cell Transformation, Neoplastic; Enzyme-Linked Immunosorbent Assay; Genes; Humans; Methylnitronitrosoguanidine; Mice; Protein-Tyrosine Kinases

In summary, two in vitro systems with rat tracheal epithelial cells were used to demonstrate that initiated respiratory tract epithelial cells can be promoted or enhanced to transform at a higher frequency by exposure to a noncarcinogenic tumor promoter. The latter two studies suggest possible mechanisms for this enhancement: 1) TPA amplifies the chromosomal instability brought about by initiating doses of carcinogen and 2) TPA alters gene expression and cellular differentiation such that it causes the initiated cell to escape the normal differentiation and senescence process thereby enhancing the proliferative lifespan of initiated cells.

Topics: Aneuploidy; Animals; Cell Transformation, Neoplastic; Cells, Cultured; DNA; Keratins; Methylnitronitrosoguanidine; Rats; Rats, Inbred F344; Tetradecanoylphorbol Acetate; Tracheal Neoplasms

Topics: Cell Transformation, Neoplastic; Cells, Cultured; Diethylstilbestrol; Endometrium; Female; gamma-Glutamyltransferase; Humans; L-Lactate Dehydrogenase; Methylnitronitrosoguanidine; Phenotype; Tetradecanoylphorbol Acetate

In a population of cultured rat liver epithelial cells transformed by 11 brief treatments with N-methyl-N'-nitro-N-nitrosoguanidine, 9% of the cells stained intensely for gamma-glutamyl transpeptidase (GGT). We have isolated from this phenotypically heterogeneous tumorigenic cell population 11 GGT-positive and 7 GGT-negative clonal subpopulations (from single cells) and have analyzed the ploidy and selected biochemical, histochemical, and growth properties of the cells in these clonal sublines. As compared to the GGT-negative strains and normal diploid rat liver epithelial cells, cells of the GGT-positive strains are larger in size, have greater DNA content, proliferate more slowly in culture, and have higher specific activities of NADH diaphorase, glucose-6-phosphate dehydrogenase, pyruvate kinase, and lactate dehydrogenase. The GGT-positive strains also show greater alteration and heterogeneity than do the GGT-negative strains in their ability to store glycogen and in their expression of lactate dehydrogenase isozymes. The results indicate that enzymatic changes commonly observed in "altered" hepatocytes in rat livers exposed to chemical carcinogens in vivo can also be produced in vitro in cultured hepatic epithelial cells by treatment with carcinogens. Moreover, treatment of a cell line with a chemical carcinogen generates a population of cells vastly heterogeneous in both their phenotypes and genotypes. Isolation of clonal subpopulations from the resulting cell line allows critical examination of the linkage and mechanistic relationship between tumorigenicity and many paratumorigenic phenotypes.

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Clone Cells; Dihydrolipoamide Dehydrogenase; Epithelium; gamma-Glutamyltransferase; Glucosephosphate Dehydrogenase; Isoenzymes; L-Lactate Dehydrogenase; Liver; Liver Neoplasms, Experimental; Methylnitronitrosoguanidine; Phenotype; Pyruvate Kinase; Rats

Clonal subpopulations of a chemically induced tumorigenic rat liver epithelial cell line were analyzed for their cellular, biochemical, and in vitro growth properties and their tumorigenicity after injection into day-old newborn isogeneic rats. The phenotypic properties studied included DNA content; growth rate in culture; activities of gamma-glutamyl transpeptidase, NADH diaphorase, pyruvate kinase, glucose-6-phosphate dehydrogenase, and lactate dehydrogenase; ability to grow in calcium-poor medium; and ability to form colonies in soft agar. The results show that none of these phenotypes cosegregates with tumorigenicity and therefore is not reliable as a "marker" phenotype for neoplastic transformation in cultured rat liver epithelial cells. The poor correlations, either qualitatively or quantitatively, between paratumorigenic phenotypes and tumorigenicity suggest that neoplastic transformation in these cells involves a specific transforming gene locus or loci and that in vitro paratumorigenic phenotypes are merely epiphenomena of neoplastic transformation and progression. This study further reveals that the efficiency of the tumorigenicity assay of cultured rat liver epithelial cells in isogeneic newborn rats can be considerably improved by incubating the cells in medium containing only trace amounts of serum prior to transplantation into the host animals.

Topics: Animals; Cell Transformation, Neoplastic; Epithelium; Fibronectins; gamma-Glutamyltransferase; Liver; Liver Neoplasms, Experimental; Methylnitronitrosoguanidine; Neoplastic Stem Cells; Phenotype; Rats

Oncogenic derivatives of Madin-Darby canine kidney (MDCK) cells were isolated in the nude mouse, and nononcogenic anchorage-independent transformants were isolated in vitro following chemical mutagenesis in vitro. These transformed cell lines as well as a Moloney sarcoma virus (MSV) transformed line were characterized with respect to their serum and anchorage requirements, growth rates, final saturation densities, and sensitivities to contact inhibition. None of these in vitro growth characteristics were found to correlate with tumorigenicity in nude mice. One tumorigenic clone, MDCK-T1, was characterized with respect to serum-free growth requirements, cAMP production, and ornithine decarboxylase (ODC) activity. These cells exhibited a significant reduction in the PGE1 requirement for growth, they produced higher levels of cAMP, and they expressed a reduced level of ODC activity relative to the parental MDCK cells. These findings may reflect changes in growth control mechanisms which accompany kidney epithelial cell tumorigenesis and suggest that the study of transformed lines derived in this manner could lead to the identification of in vitro properties which are associated with malignancy.

Topics: Animals; Cell Division; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cyclic AMP; Dogs; Epithelial Cells; Epithelium; Ethyl Methanesulfonate; Kidney; Methylnitronitrosoguanidine; Mice; Mice, Nude; Ornithine Decarboxylase

Induction of transformation, cell lethality, and DNA lesions were quantitatively compared in Syrian hamster embryo cells (HEC) treated with three different methylating agents: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), N-methyl-N-nitrosourea (MNU), or methyl methanesulfonate (MMS). Each induced transformation in a dose-dependent manner. On a molar basis, MNNG was approximately equal to 100- and 500-fold more effective than MNU and MMS, respectively. For each carcinogen the induction and repair of O6- and N7-methylguanine (O6- and N7-MeGua) relative to total guanine content was compared. At concentrations that induced equivalent transformation frequencies, the induction of O6-MeGua was the same for all three carcinogens, but N7-MeGua induction was 30-fold higher with MMS than with MNNG or MNU. The capacity to repair methylation lesions in HEC is limited because only between 50% and 70% of both O6- and N7-MeGua lesions were removed from the DNA within 24 hr after treatment, independent of methylating carcinogen. No consistent effect on either the rate of DNA replication or the size distribution of nascent strands correlated with O6-MeGua induction. These data support the hypothesis that O6-MeGua is the critical lesion for initiation of carcinogenesis by methylating agents. The frequency of transformation relative to O6-MeGua induction is 40- to 750-fold more than that of mutation. Based on the quantitative data for induction of O6-MeGua and transformation, the target size for initiation of carcinogenesis was calculated as a minimum of 10(4) nucleotides. This suggests that one of many genes can initiate carcinogenesis or that initiation is not the result of a single base mutation.

Topics: Animals; Cell Transformation, Neoplastic; Cricetinae; DNA; Dose-Response Relationship, Drug; Female; Guanine; Mesocricetus; Methyl Methanesulfonate; Methylation; Methylnitronitrosoguanidine; Methylnitrosourea; Nitrosourea Compounds; Pregnancy

Two sets of abundant cytoplasmic transformation-specific polypeptides, p788/p789 and p219/p220, have been identified by comparing in vitro-transformed human fibroblasts with diploid human fibroblasts. These polypeptides are also expressed by the human fibrosarcoma and osteosarcoma cell lines HT1080 the human fibrosarcoma and osteosarcoma cell lines HT1080 and HOS, respectively. HOS cells, however, synthesize only one of the two electrophoretic forms of each marker set, p789 and p219, at greatly reduced rates compared to the rates of synthesis found for HT1080 cells and the in vitro-transformed cell lines. Induction of expression of these neoplastic marker polypeptides is independent of the activation of a transforming gene that will induce focus formation in confluent mouse 3T3 cell monolayers. Activation of the met oncogene in MNNG-HOS cells and simultaneous elevation of tumorigenic potential did not lead to a significant change in the rate of the 600 most abundant polypeptide species with the exception of one of the two cytoplasmic actin polypeptides. While the normal ratio of beta-to gamma-actin which is approximately 2:1 was expressed in "untransformed" HOS cells, MNNG-HOS cells synthesized 50% less beta-actin resulting in a 1:1 ratio of beta-actin to gamma-actin. Our finding here, together with our previous characterization of the human beta-actin gene, leads us to predict that one of two functional beta-actin genes expressed in HOS cells has been inactivated in MNNG-HOS cells by either a regulatory or structural gene mutation.

Topics: Actins; Cell Transformation, Neoplastic; Cells, Cultured; Electrophoresis, Polyacrylamide Gel; Fibroblasts; Humans; Methylnitronitrosoguanidine; Neoplasm Proteins; Osteosarcoma

We previously have identified a subpopulation of contact-insensitive (CS-) cells which lacks density-dependent inhibition of cell division in primary and low-passage cultures of Syrian hamster embryonic (SHE) fibroblastic cells. Further, we have shown that the proportion of these CS- cells declines as a result of the stable phenotypic conversion of the CS- cells to contact-sensitive (CS+) cells. To determine whether these transient CS- cells are more sensitive to carcinogenic/mutagenic perturbation, the susceptibility to neoplastic transformation and somatic mutation induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was examined in clonally isolated cell cultures containing various proportions of CS- cells (0.02-4%). The frequencies of morphological transformation, focus formation, and neoplastic transformation showed a positive correlation to the proportion of CS- cells in the treated cultures. In contrast, the frequency of MNNG-induced somatic mutation at the Na+,K+-ATPase locus was similar among cultures varying in their proportion of CS- cells. Thus, there is a transient subpopulation of CS- cells in primary SHE cell cultures that is more susceptible to neoplastic transformation although equally susceptible to induced point mutation. This dissociation between somatic point mutation and neoplastic transformation indicates a fundamental difference in the nature of these two phenomena. A possible relationship between the propensity of CS- cells (versus CS+ cells) to carcinogen-induced neoplastic transformation and the state of differentiation of the CS- cells is discussed.

Topics: Animals; Cell Adhesion; Cell Differentiation; Cell Division; Cell Line; Cell Survival; Cell Transformation, Neoplastic; Contact Inhibition; Cricetinae; Dose-Response Relationship, Drug; Mesocricetus; Methylnitronitrosoguanidine; Mutation

The effect of epidermal growth factor (EGF) on the capacity for anchorage-independent growth of chemically treated rat hepatic epithelial cells has been investigated. We have performed the studies using 16 clonally derived cell strains which represented single cell-derived subpopulations of a heterogeneous rat hepatic epithelial cell line that had been tumorigenically transformed by 11 repeated treatments with N-methyl-N'-nitro-N-nitrosoguanidine. The results can be summarized as follows. (a) Secondary clonal subpopulations isolated from the colonies formed by these strains in soft agar subsequently and invariably acquired markedly enhanced colony-forming efficiencies as compared to their parental strains. (b) EGF could enhance or induce colony-forming ability in soft agar in all of these cell strains. (c) The magnitudes of enhancement of the colony-forming efficiencies by EGF in soft agar could not be correlated with the absolute EGF-binding capacity of these cell strains. (d) The enhancement or induction of the colony-forming ability by EGF was either reversible or irreversible, partially correlating with the expression of gamma-glutamyl transpeptidase activity by the strains. These findings indicate that the cellular capacity of liver epithelial cells to grow anchorage independently in soft agar medium can be graded according to the pattern of response of EGF induction of colony-forming ability. These grades may reflect the level of neoplastic transformation of these cells. Moreover during the multistep transformation of rat hepatic epithelial cells by a chemical carcinogen, EGF can be used to reveal the presence of altered cells which have acquired partial capacity for the anchor-age-independent growth property. This property may constitute an additional identifiable early step of the neoplastic transformation of these cells.

Topics: Animals; Cell Transformation, Neoplastic; Cells, Cultured; Epidermal Growth Factor; Liver; Liver Neoplasms, Experimental; Methylnitronitrosoguanidine; Rats

The induction of neoplastic cell transformation is closely associated with DNA alterations which occur shortly after carcinogen exposure. Sister chromatid exchange (SCE) formation is a sensitive indicator of carcinogen-DNA interaction and correlates with the induction of morphological cell transformation. The persistence of lesions generating SCE produced by chemical and physical carcinogens and its relevance to the induction of morphologic transformation was evaluated in coordinated experiments with cultured Syrian hamster fetal cells (HFC). Exponentially growing HFC were exposed for 1 h to benzo[a]pyrene (BP), methyl-methanesulfonate (MMS), cis-platinum (II) diaminedichloride (cis Pt II), N-methyl-N'-nitrosourea (MNU), mitomycin C (MMC), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), N-acetoxy-2-fluorenyl-acetamide (AcAAF) or u.v. light irradiated. Cells were incubated for 24 h with 5-bromodeoxyuridine (BrdUrd) required for SCE visualization at 1, 24 and 48 h after carcinogen exposure. The induction of morphological transformation was determined on the quantitative colony assay at 6 days after carcinogen treatment. SCE analysis demonstrates that for a period of 48 h after carcinogen exposure, during which time the cells undergo at least four replicative cycles, DNA damage generating SCE induced by all chemical carcinogens either persisted or was partially removed, whereas u.v.-induced lesions were completely removed. An elevated SCE frequency persisted after two additional cell cycles after treatment with BP, AcAAF or MMC without increased cell lethality as compared to other carcinogens whose lesions were completely eliminated during the same period. Although a correlation between the persistence of SCE and the induction of transformation was not observed for all carcinogens, this study illustrates that DNA damage generating SCE can persist over several replicative cycles, thus raising the possibility that lasting DNA alterations are important for the induction of neoplastic cell transformation.

Topics: Animals; Benzo(a)pyrene; Carcinogens; Cell Transformation, Neoplastic; Cells, Cultured; Cricetinae; DNA; Fetus; Mesocricetus; Methyl Methanesulfonate; Methylnitronitrosoguanidine; Mitomycin; Mitomycins; Sister Chromatid Exchange

Syrian golden hamster pancreatic organ explants were treated with either methylnitrosourea (MNU) or N-methyl-N-nitroso-N'-nitroguanidine (MNNG). In control explants treated with only dimethylsulfoxide, there was evidence of autophagy and crinophagy in acinar cells. Carcinogen-exposed explants showed increased numbers of autophagic and crinophagic vacuoles. In long-term cultured explants there was an increase in the number of ducts over zero time control tissues. Eosinophilic cells similar to hepatocyte-like cells were seen in 90% of the carcinogen-treated explant experiments and in 45% of the controls. Nitrosamine exposure did not induce an increase in the overall amount of necrosis measured morphometrically. Nitrosamine exposure in vitro appears to lead to a sequence of events that follow carcinogen metabolism by the acinar cells. The changes that follow include altered cell morphology and toxic cell injury evidenced by autophagic and crinophagic processes, regeneration of ductal appearing cells, the appearance of hepatocyte-like cells and an overall increase in the amount of ductal metaplasia. Within some of these ductal foci, several ductules show atypical features.

Topics: Animals; Cell Transformation, Neoplastic; Cricetinae; Mesocricetus; Metaplasia; Methylnitronitrosoguanidine; Methylnitrosourea; Nitrosourea Compounds; Organ Culture Techniques; Pancreas; Pancreatic Neoplasms; Phenotype

A diploid population of cultured rat hepatic epithelial cells that expresses the "oval" cell phenotype was exposed briefly and repetitively to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and the effect on more than 20 phenotypic properties was evaluated during the neoplastic transformation of the population. MNNG treatments of this hepatic epithelial cell population resulted in a progressively increasing phenotypic alteration and heterogeneity including changes in specific activities of several cellular enzymes and expression of isozymes, synthetic functions, and various in vitro growth properties. Changes in phenotypic expression were clustered episodically and were associated with major karyotypic changes. The development of increasing phenotypic heterogeneity preceding and accompanying tumorigenicity in cultured liver epithelial cells in vitro and the specific phenotypes that occur resemble superficially the pattern of phenotypic changes that occur in hepatocytes during chemical hepatocarcinogenesis in vivo. The results of this study provide the basis for future investigations to further elucidate the mechanistic and linkage relationship between specific pretumorigenic and paratumorigenic phenotypes and tumorigenicity.

Topics: Animals; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cells, Cultured; DNA; Epithelium; Flow Cytometry; Isoenzymes; Karyotyping; Liver; Male; Methylnitronitrosoguanidine; Neoplasm Transplantation; Phenotype; Ploidies; Rats; Rats, Inbred F344

The rate of spontaneous generation and the frequency of carcinogen-induced anchorage-independent variants of preneoplastic rat tracheal epithelial (RTE) cells in culture were quantitated. Anchorage-independent variants of different RTE cell lines arose spontaneously by a stochastic process at rates of 0.5 X 10(-4) to 5.4 X 10(-4) variants/cell/generation, as determined by fluctuation analyses. These variants were also induced by the mutagen N-methyl-N'-nitro-N-nitrosoguanidine with a frequency of approximately 10(-3) variants/surviving cell. The rates of spontaneous change and the frequencies of induction are the first reported for epithelial cells and are similar to some, although not all, rates and frequencies of change to anchorage independence for fibroblast-like cells in culture. In addition, these rates and frequencies are similar to those for mutations at some known gene loci. The induced frequency of this late change in neoplastic progression is, however, considerably lower than the frequency of induction of the initial, preneoplastic changes in RTE cells in culture (approximately 3 X 10(-2)/surviving cell). These quantitative determinations are useful in defining the mechanisms of late changes occurring during the progression of RTE cells to the neoplastic state.

Topics: Animals; Cell Transformation, Neoplastic; Cells, Cultured; Epithelium; Male; Methylnitronitrosoguanidine; Neoplastic Stem Cells; Rats; Rats, Inbred F344; Trachea

Diploid Syrian hamster embryo cells are particularly appropriate for the study of the transformation phenomenon in target cells. In vitro morphologic transformation occurs in a dose-dependent manner and is characterized by random crisscrossing and piling of cells; it correlates with tumorigenicity because individually transformed cell colonies can be isolated, cell lines can be developed, and the formation of tumors can be demonstrated after the injection of the transformed cells into either Syrian hamsters or athymic nude mice. HEC can also be used to investigate stages of carcinogenesis, initiation, and promotion. The susceptibility of normal HEC to transformation by environmental carcinogens including asbestos, bisulfite, nitrated non-carcinogenic polycyclic hydrocarbons, and X- or ultraviolet irradiation has made possible the determination of a variety of cell responses as they proceed to the neoplastic state. The initiation is usually a hereditary process involving single-hit kinetics and the transformation data indicate there is no measurable threshold response to carcinogens. The promotional aspects of transformation are readily modulated by environmental factors and have a threshold, as well as a maximal effect. The results of transformation studies using hamster cells indicate that in vitro studies are relevant to carcinogenesis and indicate that the various steps involved can be identified. Therefore, it should be possible to intervene with the various stages or steps leading to neoplasia so that cancer can be prevented.

Topics: Animals; Asbestos; Cell Transformation, Neoplastic; Cells, Cultured; Cricetinae; DNA; Embryo, Mammalian; Female; Lymphokines; Mesocricetus; Methylnitronitrosoguanidine; Polycyclic Compounds; Sulfur Dioxide; Tetradecanoylphorbol Acetate

Continuous treatment of C3H/10T1/2 cells with low concentrations (greater than or equal to 4 pM) of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) enhanced focus production in cultures pretreated with N-methyl-N'-nitro-N-nitrosoguanidine. Maximal enhancement occurred at 40 pM TCDD, a concentration 10 000-fold lower than that required to produce an optimal response with 12-O-tetradecanoylphorbol-13-acetate. Single treatments with 0.06 nM-5 microM TCDD did not transform C3H/10T1/2 cells or initiate the process of transformation in cultures subsequently exposed to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate. Promotion of transformation is thus the predominant effect of TCDD in the C3H/101/2 cell transformation system.

Topics: Animals; Cell Transformation, Neoplastic; Cells, Cultured; Dimethyl Sulfoxide; Dioxins; Dose-Response Relationship, Drug; Methylcholanthrene; Methylnitronitrosoguanidine; Mice; Polychlorinated Dibenzodioxins; Tetradecanoylphorbol Acetate

The development of transformed colonies and concomitant changes in proliferative and nonproliferative cell compartments were studied in rat tracheal epithelial (RTE) cell cultures following exposure to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Primary RTE cells were plated onto 3T3 feeder layers and treated with MNNG (0.25 micrograms/ml) or solvent. Seven days later, the feeder cells were removed to select for enhanced growth variants, which are the transformants of the RTE cell system, usually scored 5 weeks after carcinogen exposure. Most of the RTE cell colonies, which originally formed during the first 7 days of culture, disappeared within 2 weeks after feeder cell removal in control and MNNG-treated cultures. In control cultures, about 3% of the original colonies persisted, while in MNNG-treated cultures, a larger percentage (approximately 9%) of the colonies persisted. These percentages remained constant from 3 to 7 weeks. Based on colony size, cell density, and cell morphology, the persistent colonies were classified into transformed colonies (large colony size, high cell density, high nuclear:cytoplasmic ratio) and untransformed colonies (small size, low cell density, low nuclear:cytoplasmic ratio). In the MNNG-treated cultures, about 50% of all persistent colonies showed transformed morphology. Their frequency remained unchanged between 3 and 7 weeks of culture. In contrast, only 10 to 15% of the persistent colonies in control cultures showed transformed morphology at 3 weeks, but that proportion increased steadily between 3 and 7 weeks. These data suggest that, in control cultures, transformed colonies developed spontaneously as a function of time within untransformed colonies. Autoradiographic studies with [3H]thymidine showed that labeling indices in the early "normal" RTE cell colonies between Days 4 and 7 of culture were very high, ranging between 75 and 90%. In contrast, the labeling indices of persistent colonies, both those without and those with transformed morphology, were low, i.e., between 18 and 25%, indicating that a major proportion of cells was either noncycling or cycling very slowly. The relative compartment sizes of cells with stem cell characteristics and of cells with characteristics of transformed stem cells were estimated before and after transformed colonies appeared.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Animals; Cell Transformation, Neoplastic; Cells, Cultured; Epithelium; Male; Methylnitronitrosoguanidine; Precancerous Conditions; Rats; Rats, Inbred F344; Stem Cells; Thymidine; Trachea; Tracheal Neoplasms

Electron microscopic examination of the MNNG-induced gastric cancer revealed cytodifferentiation processes in it. Transformation of gastric epithelium under the effect of MNNG at the submicroscopic and cell levels is investigated. The model is shown to be adequate to human gastric cancer.

Topics: Adenocarcinoma; Animals; Cell Transformation, Neoplastic; Methylnitronitrosoguanidine; Microscopy, Electron; Rats; Stomach Neoplasms

Topics: Alkylating Agents; Animals; Cell Cycle; Cell Line; Cell Transformation, Neoplastic; Chromosome Aberrations; DNA; Embryo, Mammalian; Karyotyping; Methylnitronitrosoguanidine; Mice; Mice, Inbred BALB C; Mutation; Time Factors

The transformation of C3H/10T1/2 cells can be made to proceed through discrete stages of initiation and promotion. Studies of the effect of cell density upon focus formation in cultures treated with MNNG and TPA suggest that initiation by MNNG is due to a relatively infrequent, irreversible event induced by a single carcinogen treatment. In contrast, promotion appears to be a reversible process requiring multiple treatments with TPA over a protracted period of time. Some evidence suggests that promotion may entail the induction of phenotypic changes which impart a growth advantage to phenotypically unstable "initiated" cell populations. The actual cellular mechanism(s) for most of the phenomena observed in C3H/10T1/2 cultures have eluded precise definition and widely divergent hypotheses have been advanced to explain transformation, initiation, and promotion. Conceivably there are multiple mechanisms responsible for each of these phenomenon. Some agents may transform by a multistage mechanism whereas others may exert their effects in a more direct fashion. Some of the foci produced by promotion may be the result of simple selective processes, others the product of more complex inductive events. Variations would thus be expected between laboratories working with different protocols and agents. As demonstrated by the possible involvement of an MCA residue in transformation, it is also apparent that fundamental technical aspects of this conceptually simple cell transformation system are poorly understood. While it is natural to develop mechanistic models based on quantitative observations of transformation, a limited understanding of the basic cell culture variables which modulate both the induction and expression of transformation dictate that caution be exercised in extrapolating the significance of such models to in vivo carcinogenesis.

Topics: Animals; Cell Communication; Cell Line; Cell Transformation, Neoplastic; Cocarcinogenesis; Embryo, Mammalian; Methylcholanthrene; Methylnitronitrosoguanidine; Mice; Mice, Inbred C3H; Tetradecanoylphorbol Acetate

Reports of unusual increases in transformation frequency in low density cultures of C3H/10T1/2 cells suggest that transformation occurs via an indirect multistage mechanism. The effect of surviving cell density upon subsequent focus production was examined in C3H/10T1/2 cultures treated with acetone, 12-O-tetradecanoylphorbol-13-acetate (TPA), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), MNNG plus TPA, or 3-methylcholanthrene (MCA). Foci developed independent of cell density in 0.5 and 5.9% of all cultures exposed to acetone and TPA (0.25 micrograms/ml), respectively. Transformation after treatment with MNNG (0.5 micrograms/ml) occurred with low frequency (less than or equal to 7 X 10(-4)/surviving cell) and was enhanced by TPA. In MNNG plus TPA treated cultures containing less than or equal to 140 viable cells the fraction of dishes with foci was dependent upon the number of cells present at the time of MNNG treatment. As a result, relatively constant frequencies of focus formation were obtained (less than or equal to 6 X 10(-3) after correction for focus formation in TPA treated solvent controls). Focus frequency declined at cell densities greater than or equal to 350 cells. In contrast, treatment with MCA (1.0 microgram/ml) produced transformed foci with frequencies that varied from 3.3 X 10(-2) at the lowest density (5.5 cells) to 5.4 X 10(-4) at the highest (4400 cells). In low density cultures (5.6-56 cells), the fraction of dishes with foci was independent of the number of cells treated. Thus cell density had differential effects upon the frequency of foci produced by MCA or MNNG plus TPA. However, binding studies demonstrated that 6-7% of the MCA added to cell culture dishes was retained after the termination of carcinogen treatment. This residual MCA possessed biological activity which may be sufficient to elevate transformation frequencies in low density cultures.

Topics: Animals; Cell Line; Cell Survival; Cell Transformation, Neoplastic; Methylcholanthrene; Methylnitronitrosoguanidine; Mice; Research Design; Tetradecanoylphorbol Acetate

Topics: Animals; Azaguanine; Benzo(a)pyrene; Carcinogens; Cell Line; Cell Survival; Cell Transformation, Neoplastic; Cricetinae; Drug Resistance; Methylnitronitrosoguanidine; Mice; Mutation; Ouabain

To investigate the transforming potency of flavoring agents, Chinese hamster (CH) B241 cells were treated with various concentrations of flavoring agents for 24 hours during their exponential growth period. Surviving cells were cultivated for generations without the agents to observe the appearance of growth properties characteristic of transformed cells. Highly increased rates of structural and numerical chromosome abnormalities were seen in the cells treated with a high concentration of the agents, especially with allylisothiocyanate (AITC) or tr-cinnamic aldehyde (CA). During subsequent passages of the treated cells, the survivors from treatment with sublethal doses of AITC or CA showed a significant increase in cloning efficiency in soft agar medium, especially after passage in soft agar. Also noted was an increase in saturation density in monolayer culture, though a significant increase in plating efficiency at low serum level was not observed. These characteristic changes in AITC- or CA-treated cells were associated with the significant increase in frequencies of cells containing almost 3n to 4n chromosomes.

Topics: Animals; Cell Transformation, Neoplastic; Cells, Cultured; Chromosome Aberrations; Colony-Forming Units Assay; Cricetinae; Cricetulus; Flavoring Agents; Karyotyping; Methylnitronitrosoguanidine

Three approaches have been taken to study simultaneously Syrian hamster cells and human cells in order to develop an extrapolation from the more established hamster system to human cells. On the Characterization of normal cells in comparison to tumor cells, human tumor cells and hamster tumor cells showed similarity in displaying anchorage independence, growth in suspension as micro tumor spheroids, and xenotumorigenicity in contrast to their respective normal cells; in addition, these tumor cells exhibited shorter population doubling time, higher saturation density, higher cloning efficiency, and higher fibrinolytic activity relative to their respective normal cell types. Other differences including ploidy change, contact inhibition on growth, serum requirement, and morphological transformation were also noted between human and hamster cells. On the application of microcarrier culture for a xenotumorigenicity test, the microcarrier technique seemed to have enhanced the sensitivity by reducing the number of inoculated tumor cells required for tumor formation. On the in vitro transformation of normal human and hamster cells, the highest efficiency of morphological transformation of hamster cells has been observed in the group treated with N-methyl-N'-nitro-N-nitrosoguanidine followed by griseofulvin which was employed to enhance the transformation by disturbing the chromosome apparatus. However, no evidence of transformation was observed in the treated human cells thus far.

Topics: Animals; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cricetinae; Embryo, Mammalian; Female; Fetus; Fibroblasts; Fibrosarcoma; Griseofulvin; Humans; Kinetics; Mesocricetus; Methylnitronitrosoguanidine; Pregnancy; Species Specificity; Tetradecanoylphorbol Acetate

The biologically active metabolite of vitamin D3, 1 alpha,25-dihydroxycholecalciferol [1,25-(OH)2D3], which by itself was not effective in inducing morphological cell transformation in vitro in the Syrian hamster embryo colony assay, enhanced such a transformation in a dose-dependent manner in cells pretreated with a series of known chemical carcinogens. Treatment of the hamster embryo cells with either benzo[a]-pyrene (B[a]P), (+/-)7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, or N-methyl-N'-nitro-N-nitroso-guanidine for 3 days (stage 1) followed by treatment with 1,25-(OH)2D3 for 4 days (stage 2) increased the transformation frequency compared to the transformation frequency for cells treated with the carcinogen only. Reversing the order of the treatment (i.e., incubating the cells with 1,25-(OH)2D3 prior to B[a]P treatment) did not result in an effective enhancement. Vitamin D3 and 24,25-dihydroxycholecalciferol, another metabolite of this vitamin, also enhanced the frequency of cell transformation but to a lesser degree than did 1,25-(OH)2D3. Pyrene, which is not a carcinogen, did not induce transformed colonies either by itself or when combined with 1,25-(OH)2D3. Benzo[e]pyrene (B[e]P), which is not considered to be a complete carcinogen but can act as a tumor initiator, also was not effective by itself. However, in the two-stage protocol with 1,25-(OH)2D3, B[e]P did induce transformed colonies. Comparison of the enhancing effect of 1,25-(OH)2D3 to that of phorbol-12-myristate-13-acetate (PMA), a known tumor promoter, revealed a heterogeneity in the response to these agents. A high or low responsiveness of the cells to 1,25-(OH)2D3 was not necessarily indicative of a similar responsiveness to PMA. These results indicate that 1,25-(OH)2D3 can act as a promoter of cell transformation in fibroblasts and perhaps other cell types but through a mechanism not necessarily identical to that involving PMA.

Topics: 24,25-Dihydroxyvitamin D 3; 7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; Animals; Benzo(a)pyrene; Benzopyrenes; Calcitriol; Carcinogens; Cell Transformation, Neoplastic; Cells, Cultured; Cholecalciferol; Cricetinae; Dihydroxycholecalciferols; Embryo, Mammalian; Female; Mesocricetus; Methylnitronitrosoguanidine; Pregnancy

To define the in vitro response of nonparenchymal liver cells to chemical carcinogens, an endothelial-like cell line (RLC-1) derived from adult rat hepatic tissue was established in long-term culture. The RLC-1 line exhibited morphologic, ultrastructural and cytoskeletal characteristics consistent with derivation from mesenchymal vascular elements within the liver. Using various criteria to monitor changes in growth properties, RLC-1 cells were determined to be resistant to transformation by diethylnitrosamine, partially transformed by benzo[a]pyrene, and completely transformed by methylazoxymethanol acetate and N-methyl-N'-nitro-N-nitrosoguanidine. The RLC-1 cell system provides a means to assess potential effects of procarcinogens, as well as intrinsically reactive compounds, on an isolated nonparenchymal liver cell population.

Topics: Animals; Benzo(a)pyrene; Benzopyrenes; Cell Line; Cell Transformation, Neoplastic; Diethylnitrosamine; Flow Cytometry; Immunodiffusion; Liver; Methylazoxymethanol Acetate; Methylnitronitrosoguanidine; Rats; Rats, Inbred Strains

The intracellular levels of poly(ADP-ribose) in cultured mouse cells were increased in response to hyperthermic treatment (43 degrees C). When hyperthermia was combined with other stressful treatments such as with ethanol and/or an alkylating agent, a dramatic synergistic increase in polymer levels was observed. The effect of hyperthermia did not appear to be related to the presence of DNA strand breaks. A possible involvement of poly(ADP-ribose) metabolism in the general cellular response to environmental stress is suggested.

Topics: Animals; Cell Transformation, Neoplastic; Cells, Cultured; DNA; Ethanol; Fibroblasts; Hot Temperature; Kinetics; Methylnitronitrosoguanidine; Mice; Mice, Inbred BALB C; Nucleoside Diphosphate Sugars; Poly Adenosine Diphosphate Ribose; Simian virus 40

In order to develop a model to study cytological alterations in human pancreatic cancer, we have exposed bovine pancreatic ductal organ explants to a single dose of 2.5 micrograms per ml of N-methyl-N-nitroso-N'- nitroguanidine (MNNG). At serial time intervals, contact cytology smears of the explants were prepared and stained with the Papanicolaou stain. Control samples contained classic tall columnar and cuboidal ductal cells. Cells were uniform in size, shape, staining characteristics, and nuclear morphology. Ductal explants exposed to MNNG progressed through a series of dysplastic and atypical stages during the first 5 to 12 days in culture. Chromatin became increasingly more granular and nucleoli increased in both number and size. Exposed cells were larger than controls and had many dysplastic features. From day 15 to 30, the cells underwent changes morphologically consistent with those of human pancreatic adenocarcinomas .

Topics: Adenocarcinoma; Animals; Cattle; Cell Transformation, Neoplastic; Methylnitronitrosoguanidine; Pancreatic Ducts; Pancreatic Neoplasms; Time Factors

Highly immunogenic "tum-" (non-tumorigenic in normal syngeneic hosts) clonal variants can be selected from a variety of poorly immunogenic and highly tumorigenic mouse cell lines at very high frequencies (e.g., greater than 80%) after treatment in vitro with chemical mutagens such as ethyl methanesulfonate (EMS) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). We herein demonstrate that the same result can be obtained with the poorly mutagenic cytidine analogue, 5-azacytidine, a strong DNA hypomethylating agent. 5-Azacytidine and EMS were equally and comparably effective, or ineffective, in inducing tum- variants from three different highly tumorigenic mouse cell lines. Like mutagen-induced tum- variants, those obtained after 5-azacytidine treatment generated usually strong cytolytic T lymphocyte (CTL) responses in vitro, and could grow in immunosuppressed (nude mouse) hosts. However, pretreatment of the tumor cell lines with 5-azacytidine did not cause significant increases in mutations at several independent drug-resistant gene loci, whereas EMS did. It is known that treatment of cells with 5-azacytidine can induce transcriptional activation of "silent" genes through a reduction of DNA 5-methylcytosine content, a process that can also be effected by mutagenic DNA alkylating agents such as EMS and MNNG. We therefore hypothesize that an "epigenetic" mechanism (DNA hypomethylation) leading to activation and expression of genes coding for potential tumor antigens is involved in the generation at high frequency of tum- variants after "mutagen" treatment. The implications of these findings to mechanisms of tumor progression and the generation of tumor heterogeneity are discussed.

Topics: 5-Methylcytosine; Animals; Antigens, Neoplasm; Azacitidine; Cell Transformation, Neoplastic; Cytosine; DNA; Ethyl Methanesulfonate; Mammary Neoplasms, Experimental; Mast-Cell Sarcoma; Methylnitronitrosoguanidine; Mice; Mice, Inbred A; Mice, Inbred DBA; Mice, Nude; Mutagens; Neoplasm Transplantation; Phenotype

Chicken helper factor (chf)-negative chick embryo cells (CEC) were treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), cultured at 41 degrees in a CO2-incubator and are currently in the 400th passage (over 4 years). They were designated as CHCC-OU1. They pile up, and form colonies in soft agar, but do not produce tumors either in the syngeneic chicken or in nude mice. Chf-negative CEC without MNNG treatment were maintained in the same way and are currently in the 350th passage (over 3.5 years). They were designated as SPCC-OU1. They appear normal and form no colony in soft agar. Both cell lines produce avian endogenous leukosis virus of subgroup E.

Topics: Animals; Avian Sarcoma Viruses; Cell Line; Cell Transformation, Neoplastic; Chick Embryo; Chromosomes; Fibroblasts; Methylnitronitrosoguanidine; Phenotype; RNA-Directed DNA Polymerase; Viral Interference; Virus Cultivation

Fetal guinea pig cells were transformed by treatment with four different chemical carcinogens including nitroso compounds and polycyclic hydrocarbons. As a consequence of this treatment, oncogenes capable of transforming NIH/3T3 cells became activated in each of five independently established clonal guinea pig cell lines. Molecular characterization of representative NIH/3T3 transformants revealed that the same oncogene was present in each of the cell lines tested. Moreover, detection of this transforming gene paralleled the acquisition of tumorigenic properties by these neoplastic cells.

Topics: Animals; Base Sequence; Benzo(a)pyrene; Benzopyrenes; Carcinogens; Cell Division; Cell Line; Cell Transformation, Neoplastic; Diethylnitrosamine; DNA Restriction Enzymes; Gene Expression Regulation; Genes, Viral; Guinea Pigs; Methylcholanthrene; Methylnitronitrosoguanidine; Mice; Oncogenes; Retroviridae

Topics: Animals; Carmustine; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Colony-Forming Units Assay; DNA Repair; DNA, Single-Stranded; Drug Resistance; Fibroblasts; Humans; Lomustine; Methylnitronitrosoguanidine; Methyltransferases; O(6)-Methylguanine-DNA Methyltransferase; Simian virus 40

Dominant transforming genes that were transferred to mouse NIH3T3 cells by cellular DNAs prepared from a chemically transformed human cell line (MNNG-HOS), a human teratocarcinoma cell line (PA1), and a human pancreatic carcinoma cell line (A1165) were characterized (a) analyzing the repetitive human DNA sequences that were associated with the transforming gene and (b) determining their relationship to the oncogenes of the Harvey (rasH) and Kirsten (rasK) sarcoma viruses and to the human neuroblastoma transforming gene (rasN). The results show that the transforming gene activated in the teratocarcinoma cell line is identical to the neuroblastoma transforming gene and that the transforming gene of the pancreatic carcinoma cell line is a human homologue of rasK. In contrast, the transforming gene activated in the chemically transformed human cell line showed no detectable homology to rasK, rasH, and rasN.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Cell Line; Cell Transformation, Neoplastic; Cells, Cultured; Fibrosarcoma; Humans; Kidney Neoplasms; Leiomyosarcoma; Methylnitronitrosoguanidine; Mice; Mice, Nude; Neoplasm Transplantation; Oncogenes; Pancreatic Neoplasms; Teratoma; Transfection; Transplantation, Heterologous

Studies were carried out to examine the susceptibility of normal and initiated tracheal epithelial cells of rats to known tumour-promoting agents. The ability of normal rat tracheal epithelial cells to form colonies in cultures was enhanced markedly by addition of known tumour promoters to the medium. Several retinoids were shown to reduce the ability of these cells to form colonies in culture and to inhibit this effect of 12-O-tetradecanoylphorbol-13-acetate (TPA). A transformation assay of tracheal epithelial cells was used to study promotional effects of TPA in cultures initiated with N-methyl-N'-nitro-N-nitrosoguanidine. In this assay, four stages of transformation are recognized. TPA did not affect measurably the first two transformation stages, i.e., the development of transformed colonies (enhanced growth variants) and the 'immortalization' of enhanced growth variant-derived subcultures. However, treatment of cultures with TPA during the early post-initiation period resulted in a marked enhancement of the appearance of the third transformed phenotype, which is characterized by anchorage independence of growth. The findings of these in-vitro initiation-promotion studies paralleled, in all major respects, the results obtained in in-vivo - in-vitro studies. Tracheas exposed in vivo to initiator and promoter were shown to develop the same transformants observed in the in-vitro assay. TPA affected primarily the late anchorage-independence phenotype. Parallel tumour induction studies showed that TPA increased markedly the incidence of tracheal carcinomas following initiation with a low dose of 7,12-dimethylbenz[a]anthracene. The studies thus demonstrate that TPA is an effective tumour promoter for rat tracheal epithelium, causing an increase in tracheal carcinomas. They further suggest that the action of TPA on an early transformed cellular phenotype enhances the development of later phenotypes, including neoplastic cell variants.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinogens; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cells, Cultured; Epithelial Cells; Epithelium; Humans; Methylnitronitrosoguanidine; Mice; Rats; Respiratory Tract Neoplasms; Tetradecanoylphorbol Acetate; Trachea; Tracheal Neoplasms

We studied the effect of bile acids on the chemically induced transformation of C3H/10T1/2 fibroblasts in vitro. Bile acids exerted marked cytotoxicity on the cells at concentrations of greater than 100 microM lithocholic acid or deoxycholic acid, greater than 150 microM chenodeoxycholic acid, and greater than 500 microM cholic acid. N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) at 13.6 microM showed remarkable cytotoxicity to the cells but produced transformed cell foci in the cultures. When the cells were pretreated with MNNG and then maintained in medium containing bile acids, they showed an increased number of transformed cell foci compared to cells treated with MNNG alone. No promotion was observed in cultures treated first with bile acids and then with MNNG. On the basis of the present data, we conclude that bile acids promote the carcinogenic process of C3H/10T1/2 cells and that such promoting activity is observed not only with secondary but also with primary bile acids.

Topics: Animals; Bile Acids and Salts; Carcinogens; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Methylnitronitrosoguanidine; Mice; Mice, Inbred C3H; Structure-Activity Relationship

The effect of retinoic acid (RA) on N-methyl-N'-nitro-N-nitrosoguanidine-induced transformation of primary cultures of rat tracheal epithelial cells was investigated. RA inhibited transformation of rat tracheal epithelial cells by up to 95% at concentrations of 3.3 to 33 nM which did not substantially affect cell survival. The inhibitory effect of RA on transformation was concentration dependent and was also dependent upon timing and duration of treatment. Treatment with RA for only 1 week following N-methyl-N'-nitro-N-nitrosoguanidine exposure diminished the transformation frequency by 30 to 57%, although longer treatment times were more effective. Because RA was able to inhibit transformation effectively at concentrations which were not substantially inhibitory to colony-forming efficiency of rat tracheal epithelial cells, the mechanism of inhibition of cell transformation does not seem to be related to cytotoxic effects of RA known to occur at high RA concentrations.

Topics: Animals; Cell Transformation, Neoplastic; Cells, Cultured; Epithelial Cells; Epithelium; Male; Methylnitronitrosoguanidine; Rats; Rats, Inbred F344; Time Factors; Trachea; Tretinoin

Transformation of mouse C3H 10T1/2 cells by various alkylating carcinogens can be modulated by inhibiting poly(ADP-ribose) synthesis with a low concentration of 3-amino-benzamide, which induces no additional toxicity or reported side effects. Transformation by methylating agents was decreased by 3-aminobenzamide, whereas transformation by ethylating agents was increased. These results confirm earlier work on transformation by methylating agents, X-rays and u.v. light. Transformation by ethylating agents, however, appears to proceed by a different mechanism.

Topics: Alkylating Agents; Animals; Benzamides; Carcinogens; Cell Transformation, Neoplastic; DNA Repair; Methylation; Methylnitronitrosoguanidine; Mice; Mice, Inbred C3H; Nucleoside Diphosphate Sugars; Poly Adenosine Diphosphate Ribose

It is shown that tumour transformation of cultured mouse cells 10T1/2C3H depends on the time after the infection with virus BAV-3. The maximal number of tumours was observed 3 weeks after infection, then the frequency of tumours was reduced. MNNG modified virus-induced tumour cell transformation differently depending on the time between cell infection and treatment with the chemical agent.

Topics: Adenoviridae; Animals; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cells, Cultured; Methylnitronitrosoguanidine; Mice; Mice, Inbred C3H; Sarcoma, Experimental; Time Factors

An endoscopical study of chronological change during the carcinogenetic process of cancer of the esophagus induced by N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) in 16 male beagle dogs was performed to clarity whether or not precancerous lesions exist in the esophagus. Redness was first observed in the mucosa in the lower and/or middle portions of the esophagus. A small nodule developed in the redness, followed by a nodule. The nodule then developed into a flat polyp which finally developed into lesions such as an elevated lesions, protrusions or elevations with depressions. Histologically, esophagitis or acanthosis in the redness and acanthosis were found in most of the small nodules. With regard to the nodules, papillomatosis was observed in half of the lesions, while either acanthosis or atypical epithelial proliferation were found in most of the flat polyps. Almost all the elevated or protruding lesions were found to be carcinoma.

Topics: Animals; Biopsy; Carcinogens; Cell Transformation, Neoplastic; Dogs; Esophageal Neoplasms; Esophagoscopy; Esophagus; Male; Methylnitronitrosoguanidine

Topics: Adenocarcinoma; Animals; Cell Transformation, Neoplastic; Gastric Fundus; Male; Methylnitronitrosoguanidine; Rats; Rats, Inbred Strains; Stomach Neoplasms; Stomach Ulcer

Mutagenesis and neoplastic transformation assays on mammalian cells in culture have been extensively used for quantitative estimates of the activity of carcinogens, in spite of the limitations that such in vitro systems have when compared with in vivo systems for tumor induction. In order to assess the validity of these correlations, a series of studies was undertaken in our laboratory with the BALB/3T3 Cl A31-1-1 mouse embryo cell line. Different carcinogens were found to induce dose-dependent frequencies of transformation, including the direct-acting alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and carcinogens that were metabolically activated by these cells through different pathways (benzo[a]pyrene, 3-methylcholanthrene, aflatoxin B1, and benzidine). Their respective level of activity on a molar basis was different from that obtained in standard Salmonella + S9 mutagenesis tests. Studies currently underway indicate the possibility of lowering the serum content in the medium considerably, thereby reducing a major variable in the assay. Methods were established for the induction of ouabain-resistant (ouar) mutants in these cells. Studies were conducted by applying 30-min MNNG exposures to cells that were synchronized by serum deprivation followed by serum-induced release from growth block. While maximal induction of mutants occurred in the S phase, the transformation frequency remained constant for treatments in G1 and early or late S.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Autoradiography; Carcinogens; Cell Line; Cell Survival; Cell Transformation, Neoplastic; DNA Repair; Drug Resistance; Methylnitronitrosoguanidine; Mice; Mice, Inbred BALB C; Mutagenicity Tests; Mutation; Ouabain; Time Factors

Topics: Acetoxyacetylaminofluorene; Animals; Benzo(a)pyrene; Carcinogens; Cell Line; Cell Transformation, Neoplastic; Cisplatin; Cricetinae; Embryo, Mammalian; Mesocricetus; Methyl Methanesulfonate; Methylnitronitrosoguanidine; Sister Chromatid Exchange

Cytotoxicity, alkali-labile DNA lesions, ouabain resistance mutations, and neoplastic transformation were analyzed concurrently in the BALB/3T3 ClA31 -1-1 cell line treated with the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) for different exposure times (15, 30, 60, 90, 120, and 240 min; 24, 48, and 72 hr). The half-life of MNNG in complete medium was approximately 70 min, both without cells and with cell numbers as used in the assays for cytotoxicity (2 X 10(2) cells/60-mm dish), transformation (1 X 10(4) cells/dish), and mutation (1 X 10(5) cells/dish). The cytotoxic effect of MNNG (0.5 or 2 micrograms/ml) appeared to be completed after an exposure time between 100 and 200 min. Maximal frequency of ouabain resistance mutations, however, was reached after a much shorter treatment time (30 to 60 min). Detection of DNA damage by alkaline elution analysis showed maximal increase in single-strand breaks already after treatment for 30 min. Exposures for 30 min followed by posttreatment incubation for 30 or 90 min showed active repair of single-strand breaks during these periods, indicative of an even balance between the additional MNNG-induced damage and its repair. Morphological transformation assays, at the same treatment times and concentrations used in the mutation assays, yielded frequency curves that reached their maxima 1 to 3 hr later than did the mutation frequencies. The ratio of transformation to ouabain resistance mutation frequencies was 3.7 for short treatment times (30 to 60 min), while it increased to more than 20 for exposure times of 240 min or longer. The temporal dissociation in the exposure times for maximal induction of mutation and transformation, observed with MNNG in this cell line, supports the hypothesis that a single gene mutational event is not sufficient to account for the full expression of neoplastic transformation.

Topics: Animals; Cell Survival; Cell Transformation, Neoplastic; Cells, Cultured; Clone Cells; Drug Resistance; Kinetics; Methylnitronitrosoguanidine; Mice; Mice, Inbred BALB C; Mutation; Ouabain

The ability of eukaryotic cells in culture to proliferate in calcium-poor medium has been found to characterize populations of transformed cells, but the relationship between this phenotypic property and tumorigenicity at the cellular level is unclear. Thus, we have isolated 14 clonal subpopulations, based on their ability to colonize in calcium-poor medium, from a parental tumorigenic rat hepatic epithelial cell line which was transformed by multiple exposures to N-methyl-N'-nitro-N-nitrosoguanidine. These clonal subpopulations of cells were tested for their ability to grow in soft agar, to express gamma-glutamyl transpeptidase activity, and to form tumors upon back-transplantation into isogeneic newborn rats. The results indicated that clonal subpopulations of cells selected by their ability to grow in calcium-poor medium were phenotypically heterogeneous for gamma-glutamyl transpeptidase activity and anchorage-independent growth, and, more importantly, they were not more tumorigenic than the phenotypically heterogeneous parental cell line. This observation suggests that the capability of cultured hepatic epithelial cells to grow in calcium-poor medium is not tightly coupled to the tumorigenic phenotype.

Topics: Animals; Calcium; Cell Division; Cell Line; Cell Transformation, Neoplastic; Culture Media; Epithelium; Liver; Liver Neoplasms; Male; Methylnitronitrosoguanidine; Rats; Rats, Inbred F344

The BALB/3T3 clone A31-1-1 mouse embryo cell line at passages 7 to 13 was selected for morphologic studies of neoplastic transformation by carcinogens of different chemical classes, in the absence of any added extracellular metabolic activation. Dose-related transforming activity was demonstrated for the carcinogens aflatoxin B1 (AFB) and benzidine (BZ) not previously reported in this system, and was confirmed for benzo[a]pyrene (BP), 3-methylcholanthrene (MCA), and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Spontaneous transformation per cells at risk was low (0.14 type III foci x 10(-4), while chemically induced transformation was 2 to 3 orders of magnitude higher with all compounds. The molar concentration of carcinogens in complete medium, required to induce a transformation frequency of 1.0 type III foci x 10(-3) showed the highest level of activity for BP (0.04 microns), an intermediate level for AFB (0.2 to 1.4 microns), MCA (1.1 micron), and MNNG (2.3 microns), and the lowest level of activity for BZ (30.0 microns). The dose-related induction of morphological transformation in this clone by carcinogens of different classes indicates the potential value of this biological system in quantitative studies of carcinogen combinations, especially at low dose levels.

Topics: Aflatoxin B1; Aflatoxins; Animals; Benzidines; Benzo(a)pyrene; Benzopyrenes; Carcinogens; Cell Transformation, Neoplastic; Cells, Cultured; Clone Cells; Methylcholanthrene; Methylnitronitrosoguanidine; Mice; Mice, Inbred BALB C

After N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment of density-inhibited postconfluent (DDIR) Syrian hamster embryo cell cultures, only a small population of the cells incorporate [3H]-thymidine within the next 10-20 h. The transformed colonies, observed subsequent to reseeding, are derived from the population incorporating [3H]-thymidine. This study demonstrates that MNNG-induced thymidine incorporation resulted from semiconservative DNA synthesis as analyzed by DNA density gradients. Furthermore, cell cycle distributions at selected times after treatment indicate that the MNNG-responsive population was released from the density-dependent G1 block, proceeded through S, and became blocked again at G2 or M. These results indicate that DNA synthesis is temporally related to an early step in carcinogenesis.

Topics: Animals; Cell Transformation, Neoplastic; Cells, Cultured; Cricetinae; DNA; Embryo, Mammalian; Flow Cytometry; Interphase; Kinetics; Mesocricetus; Methylnitronitrosoguanidine; Thymidine

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

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

The tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) was found to increase the incidence of phenotypic alterations induced by the direct-acting carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in human endometrial stromal cells. Following carcinogen treatment, changes in saturation density, gamma-glutamyltranspeptidase expression, morphology, and growth in selective media were enhanced in cell cultures subjected to continuous TPA exposure as compared to cultures receiving ethanol vehicle. This enhancement may have resulted, at least in part, from selection of cells altered by carcinogen, as evidenced by differences in the colony-forming abilities of MNNG-treated and control cultures after prolonged TPA exposure, and by differences in morphologic response to TPA challenge in these two populations.

Topics: Cell Aggregation; Cell Transformation, Neoplastic; Cells, Cultured; Cocarcinogenesis; Culture Media; Female; gamma-Glutamyltransferase; Humans; Methylnitronitrosoguanidine; Phenotype; Phorbols; Tetradecanoylphorbol Acetate; Uterus

Eight cell lines exhibiting resistance to Ca2+ induced terminal differentiation were derived from primary mouse epidermal cultures and their properties analyzed. The lines developed either spontaneously (2 lines) or after exposure of primary cultures to carcinogens or carcinogens and tumor promoter. All but one of the lines were of epithelial or epitheloid morphology but 3 of the 8 lines lacked desmosomes, keratin filaments and immunoprecipitable keratin proteins, and thus could not be defined as keratinocytes. Two of the 5 keratinocyte lines were tumorigenic in syngeneic Balb/c newborns after 4 months in medium containing 1.2 mM Ca2+, and 3 lines remained non-tumorigenic even after 11 months in 1.2 mM Ca2+. All three of the non-keratinizing lines were tumorigenic. Tumorigenic potential of the 5 keratinocyte lines did not correlate with ploidy (as determined by DNA content), transglutaminase activity or growth in soft agar. However, the 2 tumorigenic keratinocyte lines contained cells which stained intensely red for gamma glutamyl transpeptidase activity, while the non-tumorigenic keratinocyte lines did not. Only those lines lacking desmosomes and keratin filaments grew in soft agar, but these lines were negative for gamma glutamyl transpeptidase activity. Ploidy and transglutaminase activity did not correlate with tumorigenicity in these non-keratinizing lines. These results show that cell lines derived from cultured mouse epidermal cells and selected on the basis of their resistance to Ca2+ induced terminal differentiation may be preneoplastic. Furthermore the association of additional markers with malignant change in these cell lines depended on whether or not the cells were keratinizing.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Benz(a)Anthracenes; Calcium; Cell Differentiation; Cell Line; Cell Transformation, Neoplastic; Drug Resistance; Keratins; Methylnitronitrosoguanidine; Mice; Microscopy, Electron, Scanning; Skin; Skin Neoplasms; Skin Physiological Phenomena

The sensitivity to sister chromatid exchange (SCE) induction by N-methyl-nitro-N'-nitrosoguanidine (MNNG) in human, mouse, Chinese hamster, and Syrian hamster normal cell strains and in permanent transformed cell lines of the same species was compared. Exponentially growing or growth-inhibited cultures of permanent cell lines transformed spontaneously or by chemical carcinogen or oncogenic virus responded with a higher SCE frequency after MNNG treatment than did normal diploid cell strains. Compared with the normals, exponentially growing Simian virus 40 transformed human fibroblast GM637 had the highest SCE frequency, followed by mouse cell line alpha L929 and Chinese hamster V79-4; the least sensitive were two Syrian hamster cell lines, OBP, derived from transformation of embryo cells with benzo(a)pyrene, and BHK, a spontaneously transformed baby hamster kidney line. Similar results were obtained with cultures arrested in G1 with glutamine-arginine deficient medium. The SCE response observed with transformed cells to carcinogen probably reflects cellular changes associated with the transformed state, such as shortening of the cell cycle, excision repair deficiency, or an increase in DNA replicon size. The current results demonstrating a difference in SCE induction between normal and malignant cells are important since normal or transformed cultured cells are utilized to assess potentially deleterious environmental agents, particularly carcinogens. In general, SCE induction by a specific direct acting carcinogen may be a useful approach for identifying transformed cells with the ability to produce tumors.

Topics: Animals; Carcinogens; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cricetinae; Cricetulus; Crossing Over, Genetic; Humans; Mesocricetus; Methylnitronitrosoguanidine; Mice; Simian virus 40; Sister Chromatid Exchange

The mutagenicity of diethylstilbestrol (DES) in V79 Chinese hamster cells was examined under a variety of conditions. DES over a concentration range 0.01-10 micrograms/ml failed to induce any increase above the spontaneous frequency of 6-thioguanine-resistant V79 cells. The effect of varying the expression time after treatment in the mutation assay from 3 to 9 days was studied and DES was nonmutagenic at all time points, while N-methyl-N'-nitro-N-nitrosoguanidine was highly mutagenic with a peak response after a 5-7 day expression time. The mutagenicity of benzo[a]pyrene and DES, both of which induce morphological and neoplastic transformation of Syrian hamster embryo (SHE) cells, was tested by cocultivating V79 cells with SHE cells for possible metabolic activation of the chemicals. Neither compound was mutagenic to V79 cells in the absence of SHE cells. Benzo[a]pyrene, but not DES, was mutagenic to V79 cells cocultivated with SHE cells. These results support the observation that DES can induce cell transformation under conditions that do not result in any measurable gene mutations. Moreover, the ability of DES to enhance the recovery of 6-thioguanine-resistant mutations was studied by determining the ability of DES to inhibit metabolic cooperation of V79 cells. Unlike the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate, DES was a weak or inactive inhibitor of metabolic cooperation.

Topics: Animals; Benzo(a)pyrene; Benzopyrenes; Cell Line; Cell Transformation, Neoplastic; Cricetinae; Cricetulus; Diethylstilbestrol; Drug Resistance; Mesocricetus; Methylnitronitrosoguanidine; Mutation; Thioguanine; Time Factors

The genetic events controlling the ability of transformed cells to grow in a medium with a low serum content (ser+) were studied. A hypodiploid clone of Chinese hamster cells with normal serum requirements (49a5ser-) was used as starting material. The results of the fluctuation tests have shown that serum-independence is a random spontaneous event. Its rate of occurrence is 1-2 . 10(-5). The concomitant study of a gene mutation (resistance to 6-mercaptopurine) revealed similar characteristics with respect to the distribution of the number of mutants in replicate cultures and the mutation rate. N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and the oncogenic SV40 virus significantly increased the frequency of ser+ colonies. In the majority of clones isolated in a medium with 1% serum (11 spontaneous and 7 induced by MNNG), the ser+ character proved to be stable after different periods of cultivation without selective pressure. The degree of serum-independence varied in different clones. The results suggest that the ability to grow in a medium with a low serum content originates, in most cases, from a mutation event.

Topics: Animals; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cells, Cultured; Clone Cells; Cricetinae; Cricetulus; Culture Media; Diploidy; Drug Resistance; Genetic Techniques; Mercaptopurine; Methylnitronitrosoguanidine; Mutation; Simian virus 40

Incubation of primary cultures of hamster embryo cells (HEC) or mouse fibroblasts (C3H/10T1/2 cells) in media depleted of thyroid hormones does not alter cell growth or survival but renders the cells resistant to neoplastic transformation by benzo[a]pyrene (B[a]P) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), carcinogens which yield transformation rates of 10(-4)-10(-2) in media supplemented with triiodothyronine (T3). In C3H/10T1/2 cells, the times of addition or removal of the hormone indicate that T3 exerts maximum effect when added 12 hr prior to treatment with B[a]P and that the progression of transformation from the time of initiation by the carcinogen to full expression and the appearance of transformed foci was independent of the presence or absence of the hormone in the medium. Dependence of transformation on T3 concentration in the medium was observed over the physiological range of 1 pM to 100 nM in C3H/10T1/2 cells treated with B[a]P. These results were similar to our previous findings on the T3 dose-related induction of radiogenic transformation and of Na+,K+-ATPase activity. The latter effect was used as a measure of T3 induction of protein synthesis. A further indication of the potential involvement of protein synthesis in T3 action is the suppression of T3- and B[a]P-dependent transformation by cycloheximide at concentrations that inhibit protein synthesis by approximately equal to 50% in the C3H/10T1/2 cells. We suggest that thyroid hormone induces the synthesis of a host protein that plays a key role in neoplastic transformation by direct-acting chemical carcinogens and by those requiring metabolic activation. In our previous studies, similar T3-dependent mechanisms were implicated in radiogenic transformations.

Topics: Animals; Benzo(a)pyrene; Benzopyrenes; Cell Transformation, Neoplastic; Cells, Cultured; Cocarcinogenesis; Methylnitronitrosoguanidine; Mice; Mice, Inbred C3H; Sodium-Potassium-Exchanging ATPase; Time Factors; Triiodothyronine

The standard C3H/10T1/2 clone 8 (C3H/10T1/2 CL8) cell transformation assay was tested for its ability to identify a variety of polycyclic hydrocarbons and alkylating agents. Dose-dependent morphologic transformation occurred with benzo[a]pyrene (BaP), 3-methylcholanthrene (MCA), 7,12-dimethylbenz[a]anthracene, BaP-7,8-dihydroxy-7,8-dihydrodiol (BaP-7,8-diol), as well as with the relatively weak in vivo carcinogen benzo[e]pyrene. Dibenz[a,h]anthracene yielded a relatively weak response, whereas anthracene and phenanthrene were negative. In contrast, treatment of C3H/10T1/2 CL8 cells with two directly acting alkylating agents, N-nitroso-N-methylnitroguanidine (MNNG) and styrene oxide, gave no transformation, whereas a third alkylating agent, ethyl methanesulfonate (EMS), gave a weak response. Treatment with MCA (2.5 micrograms/ml) yielded a reproducible positive response and, therefore, served as a positive control for routine use of the C3H/10T1/2 CL8 assay. When cells treated with the hydrocarbons BaP, BaP-7,8-diol, or MCA were analyzed for nonspecific DNA damage (single-strand breaks or alkaline-labile sites) by alkaline elution techniques, little if any DNA damage was observed. In contrast, the alkylating agents MNNG, styrene oxide, and EMS yielded substantial numbers of single-strand breaks.

Topics: Alkylating Agents; Animals; Benzo(a)pyrene; Benzopyrenes; Cell Survival; Cell Transformation, Neoplastic; Clone Cells; DNA Repair; Drug Evaluation, Preclinical; Ethyl Methanesulfonate; Methylcholanthrene; Methylnitronitrosoguanidine; Mice; Polycyclic Compounds

To study the mechanisms of carcinogenesis, we have developed a system that uses normal cells from an environmentally and epidemiologically relevant tissue, respiratory epithelium. The induction of preneoplastic variants of epithelial cells in culture was quantitated on a per-cell basis following exposure of rat tracheal epithelial (RTE) cells in vitro to the direct-acting carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Following treatment of normal RTE cells, large colonies of altered cells exhibiting an enhanced growth potential under selective culture conditions were observed, while normal RTE cells ceased proliferation after several cell doublings. After further growth in culture, these altered cells acquired the ability to grow in semisolid medium and to produce squamous cell carcinomas when injected into nude mice. The induction of enhanced growth variants of RTE cells by MNNG occurred with a high frequency (greater than or equal to 2.6%/colony-forming cell). In addition, a linear dose-response curve with a slope of approximately 1 was observed when the logarithm of MNNG-induced transformation frequency was plotted versus the logarithm of MNNG dose. These results are consistent with a one-hit mechanism for induction of preneoplastic variants of RTE cells by MNNG. Similar frequencies and kinetics of induction of preneoplastic variants in other culture systems using diploid cells have been observed, suggesting a common mechanism for this early step in carcinogenesis. The RTE cell system will be useful for mechanistic studies of early as well as late changes in the development of neoplasia by epithelial cells.

Topics: Animals; Cell Survival; Cell Transformation, Neoplastic; Cells, Cultured; Epithelial Cells; Epithelium; Genetic Variation; Kinetics; Male; Methylnitronitrosoguanidine; Precancerous Conditions; Rats; Rats, Inbred F344; Tracheal Neoplasms

Carcinogenesis is commonly considered to be a multi-step process, comprising "initiation" and "promotion" events. Skin fibroblasts from patients with hereditary retinoblastoma (RB) and familial polyposis coli (FPC) were chosen for study since their predisposition to the tumour may be due to an inherited "initiation" event which is present in every somatic cell. Thus, one might predict that skin fibroblasts from these patients might exhibit an increased susceptibility to in vitro transformation, by either tumour promoters alone or by the complete carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). In the case of skin fibroblasts from RB patients, transformation as measured by the ability of cells to grow in semi-solid medium and their migration in collagen gels, did not occur with either class of agent. However, experiments involving skin fibroblasts from FPC patients, showed these cells to grow in semi-solid medium following treatment with the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) alone, although their pattern of migration in collagen gels was unchanged and they were non-tumorigenic in nude mice. The clones which grew in semi-solid medium were also unaltered in terms of their migration in collagen gels and tumorigenicity in nude mice and were considered not to be completely transformed. These results are discussed in relation to theories that tumour promoters are only involved in cell selection and clonal expansion of initiated cells a second "mutational" event being required for complete transformation.

Topics: Carcinogens; Cell Movement; Cell Transformation, Neoplastic; Cells, Cultured; Chromosome Aberrations; Colonic Neoplasms; Eye Neoplasms; Fibroblasts; Humans; Methylnitronitrosoguanidine; Mutation; Retinoblastoma; Skin; Tetradecanoylphorbol Acetate

Primary cultures of rat tracheal epithelial cells were treated with the chemical carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) to quantitatively study the early events during neoplastic transformation. Epithelial cells were dissociated from tracheas of specific-pathogen-free Fischer-344 rats and were plated on collagen-coated tissue culture dishes. To determine cytotoxicity, cells were exposed on day 1 to various concentrations of MNNG for 3 h and colony forming efficiency (CFE) was determined on day 7. MNNG at a concentration of 0.1 microgram/ml did not decrease CFE as compared to the control cultures, whereas 1 microgram/ml reduced the CEF by 75%. For transformation studies, primary cell cultures received single exposures to MNNG (0.1-0.6 microgram/ml) or multiple exposures to 0.1 microgram/ml of MNNG for 3 h between days 1 and 17. In carcinogen-exposed cultures, morphologically altered foci appeared on day 18, recognizable by high cell density. Transformation frequencies between 1 and 8% were observed depending on MNNG concentration. Cultures containing altered foci continued to grow during the third and fourth week when control cultures had ceased to proliferate and exfoliated from the dish. Over 40% of the cultures which received multiple exposures to MNNG acquired cell line status and could be subcultured greater than or equal to 20 times. None of the 30 control cultures became cell lines. Seventy per cent of MNNG-exposed cell lines showed the anchorage independent growth phenotype at passage 20 as judged by growth in agarose. Four of 10 cultures exposed either 6 or 8 times to MNNG formed invasive squamous cell carcinomas at passage 20 upon inoculation into nude mice. Based on these and previous studies, we feel that unrestricted cell replication is an early key event in carcinogen-exposed epithelial cell populations, preceding neoplastic transformation.

Topics: Animals; Cell Transformation, Neoplastic; Cells, Cultured; Epithelium; Methylnitronitrosoguanidine; Neoplasms, Experimental; Rats; Rats, Inbred F344; Trachea; Tracheal Neoplasms

Concomitant induction of ouabain resistant mutations and morphological transformation in the BALB/3T3 ClA31-1-1-c mouse embryo cell line were obtained after a 30 min treatment with the direct alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Parameters affecting mutation frequencies were determined. The optimal expression time (48 h) for ouabain resistance was independent from the dose of the carcinogen. A linear dose-response relationship for mutation induction was found after treatment with increasing doses of MNNG. The ratio of malignant transformation to mutation frequencies induced by the short treatment with MNNG was found to be within the same order of magnitude over a four-fold dose range. The development of a mutational assay for ouabain resistance in the BALB/3T3 ClA31-1-1-c cell line makes quantitative comparisons possible between mutation and neoplastic transformation frequencies induced by chemical carcinogens in this single cellular system.

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Cells, Cultured; Clone Cells; Methylnitronitrosoguanidine; Mice; Mice, Inbred BALB C; Mutagenicity Tests; Mutagens; Mutation

C3H/10T 1/2 cells were treated with N-methyl-N'-nitro-N-nitrosoguanidine and then repeatedly exposed to formaldehyde (0.1 to 2.0 micrograms/ml). Exposure of N-methyl-N'-nitro-N-nitrosoguanidine-initiated cultures to formaldehyde concentrations of 0.5 or 1.0 micrograms/ml in a variety of treatment regimens resulted in focus formation in up to 9% of the treated dishes. Transformed foci were observed in 2% or less of the cultures treated with N-methyl-N'-nitro-N-nitrosoguanidine or formaldehyde alone. Formaldehyde thus appears to be only a weak tumor promoter for C3H/10T 1/2 cell transformation.

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Cocarcinogenesis; Embryo, Mammalian; Formaldehyde; Methylcholanthrene; Methylnitronitrosoguanidine; Mice

To determine if abnormal cellular DNA content, suggestive of aneuploidy, is an early indicator of transformation by chemical carcinogens, we exposed primary cultures of rat tracheal epithelial cells to N-methyl-N' -nitro-N-nitrosoguanidine (MNNG), 12-O-tetradecanoylphorbol-13-acetate (TPA), MNNG followed by TPA, or solvent as a control. After 40 and 60 days in culture suspensions of the cells were made, fixed, stained with DNA-dye Hoechst 33342, and the fluorescence per cell measured with a flow cytometer. The DNA histogram showed that freshly isolated rat tracheal epithelial cells, and control cells at days 40 and 60 had predominantly diploid DNA content values. The late primary (day 40) and early passaged (day 60) control cells commonly show increased numbers of cells with tetraploid DNA contents. TPA induced aneuploidy in few cultures by day 40, but by day 60 all samples tested had significant numbers of cells with aneuploid DNA content. In contrast a single MNNG treatment or MNNG followed by TPA regularly caused extensive aneuploidization by day 40. Multiple cycling subpopulations with aberrant DNA contents appear. These dramatic changes in cellular DNA content suggestive of drastic ploidy changes in MNNG and MNNG + TPA exposed cultures are early events, preceding other evidence of neoplastic transformation by many cell generations. Our results suggest that aneuploidy is an early event in the transformation of rat tracheal epithelial cell cultures by chemical carcinogens.

Topics: Aneuploidy; Animals; Cell Transformation, Neoplastic; DNA; Interphase; Male; Methylnitronitrosoguanidine; Rats; Rats, Inbred F344; Tetradecanoylphorbol Acetate; Trachea

Treatment of low density asynchronous cultures of C3H/10T1/2 Cl 8 mouse embryo fibroblasts with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) initiates the process of transformation and produces significant numbers of transformed foci only when treated cultures are subsequently exposed to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Cell culture variables which influence the outcome of this initiation and promotion system were studied. A TPA concentration of 0.25 micrograms/ml was found to be optimal for the promotion of focus production and the presence of TPA was required both during logarithmic growth and throughout confluence. The lot of fetal calf serum used to cultivate the cells also played a determining role in focus production. Of nine serum lots purchased from four different suppliers, only two were suited for initiation and promotion studies with MNNG and TPA. In contrast, seven of these lots were adequate for transformation studies with 3-methylcholanthrene. Factors which adversely influenced focus production included the use of fungizone or the use of high passage stock cultures. These studies demonstrate that cell culture variables which influence promotion in these cells can be controlled and that this system can be successfully used in studies of the cellular mechanism of in vitro promotion and for the detection of genotoxic substances.

Topics: Animals; Cell Transformation, Neoplastic; Cells, Cultured; Female; Fibroblasts; Methylnitronitrosoguanidine; Mice; Mice, Inbred C3H; Phorbols; Pregnancy; Tetradecanoylphorbol Acetate; Time Factors

Adenomatous polyps and adenocarcinomas with variety of the precancerous stages have been induced by intraectally injection of N-methyl-N'-nitro-N-nitrosoguanidine. The aim of our experiments have been to study some quantitative and qualitative changes in mucus secretion both in the neoplasm and in transitional area surrounding tumor. The changes in composition of mucus confirmed by several histochemical methods reflect the earliest morphologically observable stage of malignant transformation in colonic mucosa.

Topics: Animals; Cell Transformation, Neoplastic; Colon; Colonic Neoplasms; Methylnitronitrosoguanidine; Mucus; Precancerous Conditions; Rats; Rats, Inbred Strains

Sera obtained from rats showing transplantation immunity to syngeneic, malignant epithelial cell line, 2-10-1, were used to identify antigens associated with transformation in vitro. The 2-10-1 cell line was derived from exposure of tracheal explants, in vitro, to the carcinogen MNNG. Tumorigenic passages of the cell line were shown to have common antigenic determinants, shared by independently transformed malignant tracheal epithelial cell lines, as well as antigenic determinants that appear to be limited to the immunizing cell line, 2-10-1. Care was taken to ensure that antibodies were not produced against viral antigens or non-specifically absorbed serum components. Early passages of the 2-10-1 cell line were not tumorigenic in athymic BALB/c (nu/nu) mice, but became malignant with serial passage in vitro. Antigenic specificities recognized by 2-10-1 immune sera were also present on early-passage 2-10-1 cells that had not as yet acquired the malignant phenotype. Such antigen expression must be an early event in neoplastic development.

Topics: Animals; Antigens, Neoplasm; Cell Line; Cell Transformation, Neoplastic; Cross Reactions; Epitopes; Fluorescent Antibody Technique; Immune Sera; In Vitro Techniques; Methylnitronitrosoguanidine; Mice; Mice, Inbred BALB C; Neoplasm Transplantation; Rats; Rats, Inbred F344; Trachea; Transplantation Immunology

Oncogenic transformation of C3H10T1/2CL8 cells was improved by treating the cells 5 days after seeding. Benzo[a]-pyrene-induced transformation was increased 3.5-fold by this method, compared with treating the cells 1 day after seeding. N-Methyl-N'-nitro-N-nitrosoguanidine, which does not transform asynchronous cultures of C3H10T1/2CL8 cells when administered 1 day after seeding, produced on average of 1 focus/dish, with 61% of the dishes exhibiting foci, when administered 5 days after seeding. Propane sultone and aflatoxin B1 also produced marked transformation responses when administered 5 days after seeding. However, 4-dimethylaminoazobenzene did not induce transformation when administered either 1 day or 5 days after seeding. With all chemicals examined, clonal cytotoxicity was reduced when they were administered 5 days after seeding. These results indicate the utility of this new procedure for the qualitative analysis of the transforming ability of chemicals.

Topics: Aflatoxin B1; Aflatoxins; Animals; Benzo(a)pyrene; Benzopyrenes; Carcinogens; Cell Line; Cell Transformation, Neoplastic; Methylnitronitrosoguanidine; Mice; Probability

Twenty-one anchorage-independent subclones and ten subclones with reduced serum requirements were isolated as single-step mutants spontaneously or after ethylmethanesulfonate or N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis of CHEF/18 diploid Chinese hamster embryo fibroblasts. Anchorage-independent mutants retain the high serum requirement and nontransformed morphology typical of CHEF/18. Only four of 21 anchorage mutants have spontaneously produced tumors when injected at 10(7)/site in nude mice, and these were only at a fraction of sites. Low-serum (LS) mutants acquire transformed morphology and increased anchorage-independent growth simultaneously with the loss of high-serum requirement. Only two of ten LS mutants have spontaneously produced tumors. However, when some anchorage and LS mutants were remutagenized and when mutagenized populations were injected into nude mice, tumors appeared at many of the injected sites. In contrast, untreated CHEF/18 cells have never given tumors(0 of 34 sites), and mutagenized DHEF/18 cells have given tumors at only three of 29 sites. These results demonstrate that malignant transformation is a multistep process in the Chinese hamster embryo fibroblast system. Most one-step mutants selected for anchorage independence or reduced serum requirements do not have tumor-forming potentials higher than that of the parent CHEF/18. Thus anchorage-independent and LS phenotypes per se do not account for the increase in tumor-forming potential. It is proposed that the genomic rearrangement process as well as specific mutations may contribute to tumorigenicity.

Topics: 4-Nitroquinoline-1-oxide; Animals; Cell Line; Cell Transformation, Neoplastic; Cricetinae; Cricetulus; Culture Media; Embryo, Mammalian; Ethyl Methanesulfonate; Methylnitronitrosoguanidine; Mice; Mice, Inbred BALB C; Mice, Nude; Mutation; Neoplasms, Experimental; Phenotype; Whole-Body Irradiation

Skin fibroblasts from patients with familial adenomatosis coli (AC) and normal individuals were treated once with the carcinogen 4-nitroquinoline 1-oxide or N-methyl-N'-nitro-N-nitrosoguanidine and then passaged sequentially. Morphologically altered cells appeared in the cultures of carcinogen-treated AC fibroblasts at passages 6-8 (days 100-140) after treatment with the carcinogens, but carcinogen-treated normal cells and untreated AC and normal cells did not become altered even after cultivation for 25 passages. The cultures containing morphologically altered cells showed characteristics of transformed cells, such as a high frequency of colony formation in soft agarose, increased growth ability, and chromosomal abnormalities. The results suggest tha AC patients have increased susceptibility to morphologic transformation and chromosomal changes induced by chemical carcinogens.

Topics: 4-Nitroquinoline-1-oxide; Adenoma; Cell Count; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Chromosome Aberrations; Colonic Neoplasms; Female; Humans; Methylnitronitrosoguanidine; Middle Aged; Skin

The utility of C3/H/10T1/2 mouse embryo fibroblasts for the detection of carcinogenic substances has been limited by their apparent insensitivity to the oncogenic effects of direct-acting alkylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and procarcinogens such as aflatoxin B1 (AFB1). Because the process of C3H/10T1/2 transformation can be observed to proceed through discrete stages of initiation and promotion, we have considered the possibility that MNNG and AfB1 may only initiate C3H/10T1/2 transformation. Treatment of asynchronous C3H/10T1/2 cells with MNNG or AfB1 alone generally produced few transformed foci. If MNNG or AfB1 treatment was followed by the exposure of cells to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), numerous transformed foci were produced. Phorbol did not enhance transformation by either substance. MNNG and AfB1 thus appear to be initiating agents for transformation. TPA also enhanced the transformation of C3H/10T1/2 cells by low doses of 3-methylcholanthrene (3-MCA), but transformation by high concentrations of 3-MCA was inhibited by the presence of TPA. These studied suggest that the sensitivity of the C3H/10T1/2 transformation system to potential carcinogens can be dramatically heightened if the bioassay is conducted in the presence and absence of TPA.

Topics: Aflatoxin B1; Aflatoxins; Animals; Cell Line; Cell Transformation, Neoplastic; Methylcholanthrene; Methylnitronitrosoguanidine; Mice; Tetradecanoylphorbol Acetate

The growth responses of hamster dermal fibroblasts (HDF cells) to platelet-derived growth factor (PDGF) during chemical carcinogenesis in vitro were investigated. Normal HDF cell grew in medium with whole blood serum (WBS), but not in medium with plasma-derived serum (PDS). However, they grew in PDS medium when PDGF was added. In contrast to control cultures which finally stopped proliferating, 7 out of 8 cell strains treated with 4-nitroquinoline-1-oxide (4NQO) or N-methyl-N'-nitro-nitrosoguanidine changed to cell lines. Later, 5 of these 7 cell lines became able to grow in PDS medium in association with ability to grow in soft agar. When clonal cell lines were isolated at early stages of carcinogenesis while parental cell lines were still PDGF-dependent, most of them gradually became PDGF-independent as well. Dialysed cell lysates of transformed HDF cells showed strong growth stimulating activity on normal HDF cells in PDS medium. Thus, this conversion of PDGF-dependent cells to PDGF-independent cells was correlated with the appearance of a growth factor(s) produced specifically by transformed HDF cells.

Topics: 4-Nitroquinoline-1-oxide; Animals; Cell Line; Cell Transformation, Neoplastic; Cricetinae; Culture Media; Fibroblasts; Growth Substances; Mesocricetus; Methylnitronitrosoguanidine; Mitogens; Peptides; Platelet-Derived Growth Factor

Using repeated cloning and treatment with cis-HPL (200 micrograms/ml), an analogue of a procollagen precursor inhibitory to the growth of collagen-synthesizing cells of mesenchymal origin, clonally premature epithelial cell lines were isolated from fetal SGH lungs cultured on the 15th day of gestation. One of the cell lines, M3E3/C3, which has been extensively studied for biological characterization, developed poorly differentiated carcinomas in injected hamsters after transformation by MNNG. Moreover, when grown on collagen gel, this cell line indicated an obvious potency for in vitro differentiation in response to vitamin A by developing activated Golgi regions, well developed rER and a number of mucus-like granules. Since such a differentiative responses is expected to be definable in the light of respiratory epithelium developing in utero, this cell line may be useful for studying mechanisms of differentiation-dependent sensitivity of fetal organs to transplacental carcinogen exposure.

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Cricetinae; Epithelial Cells; Female; Fetus; Hydroxyproline; Lung; Lung Neoplasms; Maternal-Fetal Exchange; Mesocricetus; Methylnitronitrosoguanidine; Pregnancy

The transformable mouse embryo fibroblast cell line C3H10T 1/2 C18 has been employed to study the induction by the carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) of morphological transformation and mutation to ouabain resistance throughout the cell cycle. Cells were synchronized by means of isoleucine deprivation for 24 hr and initiated DNA synthesis with a high degree of synchrony 7.5 hr after release of the isoleucine block. At various intervals throughout the cell cycle cultures were treated with MNNG at 1.0 microgram/ml and the induction of cytotoxicity, morphological transformation, and ouabain-resistant colonies was determined. All three phenomena exhibited marked cell-cycle phase dependency. Maximal induction of transformation occurred in cultured treated 7.5 hr after release from isoleucine deprivation, when the cells were at the G1/S boundary. In contrast, induction of ouabain-resistant colonies was at a minimum at the time of maximal induction of transformation, and peak induction of ouabain resistance did not occur until 16-18 hr after release from the isoleucine block, when cells were in late S phase. A close correlation was observed between the induction of cytotoxicity and of ouabain-resistant mutants. The results suggest that differences exist in the production or cellular processing of the various early lesions.

Topics: Animals; Cell Cycle; Cell Line; Cell Survival; Cell Transformation, Neoplastic; Drug Resistance; Methylnitronitrosoguanidine; Mice; Mutation; Ouabain; Time Factors

Relationships among carcinogen-induced morphlogic transformation, anchorage independence, susceptibility to growth inhibition by the immunologic hormone lymphotoxin and to natural killer cell and natural lymphoid cell cytotoxicity, natural delayed-type (tuberculin) skin reactivity, and tumorigenicity were investigated with the use of a guinea pig cell culture model of carcinogenesis characterized by sequential, extended developmental stages. Twelve cultures of passage 90 nontumorigenic fibroblast-like line 118 cells derived from a 36-day-old strain 2/N guinea pig fetus were treated with 10 micrograms benzo[a]pyrene or 1 microgram N-nitroso-N-methylnitroguanidine/ml medium for 3 days and maintained in exponential growth. One week later, all cultures exhibited morphologic alteration, and by 8 weeks two-thirds exhibited morphologic transformation. After 4 months, morphologically altered or transformed cells expressed progrssive colony growth in semisolid 0.35% agar medium. Susceptibility to lymphotoxin colony inhibitory activity developed subsequent to morphologic transformation but independently of growth in semisolid medium. Susceptibility to natural lymphoid cell destruction was also independent of growth in semisolid medium. All four phenotypic properties were expressed only after carcinogen treatment and 12 months later remained unaccompanied by either natural delayed-type skin reactivity or by the ability of the cells to grow as tumors in syngeneic guinea pigs of nu/nu mude mice. Thus while morphologic transformation, colony formation in semisolid medium, and susceptibility to lymphotoxin and to nautral lymphoid cell cytotoxicity were frequently associated with neoplasia, they could be expressed independently of one another; individually or collectively in a specific developmental sequence, they were insufficient for expression of either natural delayed-type skin reactivity of tumorigenicity.

Topics: Animals; Benzopyrenes; Cell Line; Cell Transformation, Neoplastic; Colony-Forming Units Assay; Guinea Pigs; Male; Methylnitronitrosoguanidine; Mice; Mice, Nude; Neoplasm Transplantation; Neoplasms, Experimental; Phenotype

The cyclic nucleotides, cyclic adenosine 3':5'-monophosphate (cAMP) and cyclic guanosine 3':5'-monophosphate (cGMP) or their dibutyryl and monobrominated derivatives, may either increase or decrease morphological transformation of Syrian hamster embryo cells exposed to N-methyl-N'-nitro-N'-nitrosoguanidine (MNNG). The effect on transformation is primarily a function of the parent cyclic nucleotide and the duration of exposure to the nucleotides. At concentrations of 5 mM or larger for a minimum 24-hr exposure cAMP, cGMP, and their congeners reduced the colony-forming ability of nontransformed Syrian hamster embryo cells not exposed to MNNG; however, cGMP and its derivatives caused less toxicity than cAMP or its analogs. cAMP and its monobrominated and dibutyrylated derivatives decreased the transformation frequency associated with MNNG irrespective of whether the various adenylnucleotides were administered before or after MNNG. The greatest inhibitory effect of MNNG-induced transformation was obtained with N6,O2-dibutyryl cyclic adenosine 3':5'-monophosphate followed in order by 8-bromocyclic adenosine 3':5'-monophosphate and cAMP. At equimolar doses, the dibutyryl and brominated analogs of cGMP but not unsubstituted cGMP enhanced transformation when administered prior to exposure of the Syrian hamster embryo cells to MNNG but reduced the transformation frequency when added after MNNG. The enhancing and inhibitory effects of the guanine cyclic nucleotide-induced alteration of MNNG-associated transformation frequencies were dose and time dependent and occurred in the order N6,O2-dibutyryl cyclic guanosine 3':5'-monophosphate greater than 8-bromocyclic guanosine 3':5-'monophosphate much greater than cGMP. Butyric acid neither diminished nor increased MNNG-induced transformation frequency. The latter suggests that butyrate ions formed by metabolism of the cyclic nucleotide analogs were not a factor in the observed alterations of transformation frequency.

Topics: Animals; Cell Survival; Cell Transformation, Neoplastic; Cells, Cultured; Cricetinae; Embryo, Mammalian; Mesocricetus; Methylnitronitrosoguanidine; Nucleotides, Cyclic

Topics: Animals; Antipain; Caffeine; Carcinogens; Cell Transformation, Neoplastic; Cells, Cultured; Cocarcinogenesis; Cricetinae; DNA Repair; Lymphokines; Methylnitronitrosoguanidine; Mutation; Sister Chromatid Exchange; Tetradecanoylphorbol Acetate; Ultraviolet Rays; X-Rays

To provide information on the number of steps involved in preneoplastic progression in vitro, the induced and spontaneous appearances of anchorage-independent and tumorigenic variants were studied with clones of a permanent cell line of morphologically transformed, anchorage-dependent, non-tumorigenic pseudodiploid mouse cells (CAK). Tumorigenicity was assayed in the nude mouse by s.c. coinoculation of CAK cell derivatives with 2 x 10(6) human fibroblasts. Under the assay conditions, as few as 10 tumorigenic cells formed tumors. The assay permitted detection of tumorigenic variants soon after their origin and reduced the spontaneous development of new variants that result from extensive proliferation of clonal cell populations prior to testing for tumorigenicity. Anchorage-independent, nontumorigenic variants of CAK cells originated spontaneously at an estimated rate of about 10(-4)/cell/generation. Tumorigenic variants appeared spontaneously during proliferation of an anchorage-independent cell clone at an estimated rate of about 10(-7)/cell/generation but were undetectable among anchorage-dependent CAK cells. In contrast, N-methyl-N'-nitro-N-nitrosoguanidine treatment induced the appearance of tumorigenic variants in both anchorage-dependent and -independent clones with an estimated frequency of about 10(-4)/surviving clone former, which was similar to the induced frequency of ouabain-resistant variants in the same cells. Anchorage independence was expressed without tumorigenicity in new anchorage-independent variants but tumorigenic cells were always anchorage independent. We propose that CAK cells can become tumorigenic by a three-step pathway that includes changes causing morphological transformation, anchorage independence, and tumorigenicity. Our evidence is also consistent with an alternative two-step pathway where anchorage independence and tumorigenicity are acquired in a single step, since anchorage-independent, tumorigenic clones were derived from anchorage-dependent cells soon after a single mutagenic treatment.

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Clone Cells; Diploidy; Embryo, Mammalian; Metaphase; Methylnitronitrosoguanidine; Mice; Mice, Inbred Strains; Phenotype

N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) has been reported to induce BHK-21/Cl 13 cell growth in agar suspension. To determine if MNNG was also mutagenic to BHK cells, an ouabain-resistance mutation assay was established using these cells. In this system MNNG was compared to nitrosocimetidine (NC). MNNG and NC did induce ouabain-resistant mutations in BHK cells. The ability of the test compounds to methylate DNA in BHK cells was also determined, and both MNNG and NC yielded detectable levels of 7-methylguanine in treated cells. MNNG and NC were tested for the ability to transform BHK cells, and did. NC was found to be as effective a mutagen and transforming agent in BHK cells as MNNG when administered at equitoxic concentrations; approx. 4-fold less effective at equimolar concentrations.

Topics: Animals; Cell Line; Cell Survival; Cell Transformation, Neoplastic; Cimetidine; Cricetinae; DNA; Guanidines; Guanine; Kidney; Mesocricetus; Methylnitronitrosoguanidine; Mutagenicity Tests; Mutagens

A line of normal rat embryo fibroblasts was transformed with N-methyl-N'-nitro-N-nitrosoguanidine (a chemical carcinogen), SV40 and polyoma virus (two DNA viruses), and Rous sarcoma virus (an RNA tumor virus). In this study, we report a comparison of the levels of collagen synthesis and procollagen messenger RNA (mRNA) in 13 lines selected after transformation with one of these agents. Collagen synthesis and procollagen mRNA levels were compared with the degree of transformation determined from morphology, saturation density, growth in agarose, and tumorigenicity in nude mice. Each class of transformants had a characteristic level of collagen synthesis; this level correlated inversely with the degree of transformation of the rat embryo fibroblasts. In N-methyl-N'-nitro-N-nitrosoguanidine and SV40 transformants which were moderately transformed, collagen synthesis was hardly affected, but, in polyoma virus and Rous sarcoma virus transformants which were more severely transformed, collagen synthesis was 30 to 48% and 12 to 25%, respectively, of control levels. Type I procollagen mRNA activity measured in RNA from nine of the lines by an in vitro translation assay also decreased with increasing severity of transformation. Procollagen mRNA levels were reduced to about one-half of control levels in one SV40 transformant and to 17 to 23% of controls in polyoma virus and Rous sarcoma virus transformants. We conclude that, in this series of rat fibroblast lines, transformation with different agents resulted in characteristic levels of collagen synthesis and that collagen synthesis was most reduced in the cells which were most transformed by other criteria.

Topics: Animals; Avian Sarcoma Viruses; Cell Transformation, Neoplastic; Cell Transformation, Viral; Collagen; Methylnitronitrosoguanidine; Polyomavirus; Procollagen; Proline; Rats; RNA, Messenger; Simian virus 40

The immunogenicity of inbred strain 2/N guinea pig fibroblasts transformed to the malignant state in vitro by chemical carcinogens was evaluated with the use of a variety of in vivo and in vitro methods including delayed-type hypersensitivity skin and tumor transplantation tests and analysis of antibody production by immunofluorescence, complement fixation, and staphylococcal protein A binding tests. Neoplastic transformation was induced by direct treatment of cells in culture with benzo[a]pyrene, 3-methylcholanthrene, or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or by the host-mediated method by which fetuses were exposed to diethylnitrosamine or MNNG in vivo prior to cell culture. Rabbits and syngeneic guinea pigs were inoculated with unirradiated and X-irradiated clonally derived cells. Delayed hypersensitivity skin reactions to immunizing or other cells were equivalent in immunized or control guinea pigs, and no protection to tumor outgrowth from a challenge inoculum of immunizing cells was observed. Antibody activity induced in the sera of immunized guinea pigs was cross-reactive and removed by absorption with nontumorigenic cells. Rabbit antisera after absorption with fetal guinea pig cells were nonreactive with the specific immunizing or other culture cells. Chemical carcinogen-induced neoplastic transformation of guinea pig cells can, therefore, occur without formation of detectable, individually distinct cell surface tumor-specific neoantigens.

Topics: Animals; Antibody Formation; Antigens, Neoplasm; Benzo(a)pyrene; Benzopyrenes; Cell Transformation, Neoplastic; Clone Cells; Complement Fixation Tests; Fibroblasts; Fluorescent Antibody Technique; Guinea Pigs; Immunity, Cellular; Immunoglobulin G; Male; Methylcholanthrene; Methylnitronitrosoguanidine; Neoplasm Transplantation; Rabbits; Staphylococcal Protein A

Sister chromatid exchange (SCE) and morphological transformation induced by five chemical carcinogens, N-acetoxy-2-fluorenyl-acetamide (AcAAF), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methyl methanesulfonate (MMS), benzo[a]-pyrene (BP), and cisplatinum(II) diamine dichloride (PT) as well as by X-irradiation were quantified in parallel experiments with cultures of Syrian hamster embryo cells (HEC). Incubation of HEC with 5-bromodeoxyuridine (10(-5)M) for two rounds of replication (24 h) required for SCE visualization neither caused morphological transformation nor altered the transformation frequency induced by carcinogen alone. All chemical carcinogens, but not X-irradiation, produced a dose-dependent increase in SCE and transformation frequency, demonstrating the sensitivity of both assays to carcinogens. The ratios of induced SCE relative to transformation frequency, however, varied with the carcinogen. BP, MMNG, and AcAAF were similarly efficient in inducing SCE compared to transformation but were considerably less effective than MMS and PT. X-irradiation at doses of 200, 300, and 500 R did not cause transformation and induced a low frequency of SCE. On a molar basis, MMS and PT were the most effective in SCE induction relative to transformation, MNNG and AcAAF were less effective, and BP was the least effective carcinogen. The positive linear correlation between chemical carcinogen-induced SCE and transformation suggests a relationship between the two cellular responses.

Topics: Acetoxyacetylaminofluorene; Animals; Benzopyrenes; Carcinogens; Cell Transformation, Neoplastic; Cells, Cultured; Cisplatin; Cricetinae; Crossing Over, Genetic; Dose-Response Relationship, Drug; In Vitro Techniques; Mesocricetus; Methyl Methanesulfonate; Methylnitronitrosoguanidine; Neoplasms, Radiation-Induced; Sister Chromatid Exchange

The loss of N-methyl-N-nitro-N-nitrosoguanidine (MNNG)-induced adducts from DNA was quantitated during the G1 and S phases of synchronously proliferating mouse 10T1/2 cells exposed to MNNG (2 microgram/ml) when they were in either confluence-induced arrest of proliferation or at the G2-S border. When treated at either time, N-7-methylguanine and O6-methylguanine were not excised from the template DNA during the subsequent S phase. However, both lesions were efficiently removed during the G1 phase immediately following exposure to MNNG, as well as during the second G2 phase after an S phase during which no loss occurrred. In contrast, N-3-methyladenine was lost rapidly during both the G1 and S phases following MNNG treatment. N-7 Methylguanine and O6-methylguanine were removed from logarithmically growing cell populations more slowly than from cells passing synchronously through the G1 phase. However, when the observed rate of loss in logarithmic cultures was corrected for the fraction of the logarithmic population located in the S phase, the rates of loss of the two methylated bases were the same as those observed in the G1 phase in synchronous cultures.

Topics: Animals; Base Composition; Cell Division; Cell Line; Cell Transformation, Neoplastic; DNA; DNA Replication; Interphase; Kinetics; Methylation; Methylnitronitrosoguanidine; Mice; Time Factors

We have demonstrated a dose-dependent increase in the frequency of diploid human cells capable of anchorage-independent (AI) growth after treatment with the carcinogen propane sultone, followed by exponential growth to allow full expression of this phenotype (8 to 13 population doublings). Exposure to these same concentrations of propane sultone also resulted in a dose-dependent increase in the frequency of 6-thioguanine-resistant cells in the population. Procedures such as synchronization of cells and treatment just after the onset of DNA synthesis or the use of special selective medium were not essential for this induction. A very low frequency of cells with the AI phenotype was found in the control population (background). Cells which exhibited the AI phenotype spontaneously or after carcinogen treatment retained the characteristic over as many generations as tested (greater than 13). The data suggest that AI growth is the result of a mutational event.

Topics: Anthralin; Cell Adhesion; Cell Cycle; Cell Division; Cell Transformation, Neoplastic; Drug Resistance; Fibroblasts; Humans; Methylnitronitrosoguanidine; Mutation; Neoplasms, Experimental; Thioguanine; Thiophenes

The selection of Syrian hamster epidermal cells which do not terminally differentiate has provided a quantitative focus assay for in vitro chemical transformation. One-day-old Syrian hamster epidermal cells plated at 5 x 10(6)/100-mm dish were treated for 5 hr with various concentrations of N-methyl-N'-nitro-N-nitrosoguanidine. After 4 weeks, the normal epidermal cells began to terminally differentiate to keratinized squamous cells and died, but transformed epidermal colonies grew to higher cells densities and appeared as darker areas against a lightly stained normal cell background. Transformed epidermal foci were isolated and subcultured for at least 15 passages, whereas normal epidermal cells could not be subcultured under the same conditions. The transformed cells assumed the typical cobblestone-like morphology of epithelial cells, retained desmosomes and tonofilaments, and were able to use citrulline in place of arginine. Argininosuccinate synthetase (EC 6.3.4.5) activity was significantly higher in the epidermal cells than in fibroblasts. The injection of 5 x 10(6) cells of two transformed epidermal cell lines into athymic nude mice resulted in the formation of tumors which were identified as keratinizing squamous carcinomas.

Topics: Animals; Arginine; Argininosuccinate Synthase; Cell Transformation, Neoplastic; Cells, Cultured; Clone Cells; Cricetinae; Epidermis; Fibroblasts; Mesocricetus; Methylnitronitrosoguanidine; Phenotype; Pronase

Two cell lines (2-10-1 and 8-10-2) derived by exposure to primary tracheal explants to MNNG in vitro were not tumorigenic in syngeneic F-344 rats or athymic BALB/c (nu/nu) mice at early passage, but became tumorigenic at late passage. These cell lines are therefore suited to study the expression of neoantigens during neoplastic development. Transplantation resistance to late-passage, tumorigenic cells was indicated in syngeneic rats using an immunization protocol of repeated cell inoculation and tumour ablation. Spleen cells from such animals were reactive in 20h microcytotoxicity assays against neoplastic cell lines, but unreactive to normal tracheal epithelial cells. Similarly, immune spleen cells co-cultivated in vitro for 6 days with irradiated neoplastic cell lines before assay for microcytotoxicity were strongly reactive, whereas co-cultivation with normal epithelial cells did not stimulate reactivity. Antibody to these neoplastic cell lines was demonstrated in sera of tumour-resistant rats by an indirect radiolabelled-antibody binding test and by indirect immunofluorescence. There was no significant binding to normal tracheal epithelial cell outgrowths.

Topics: Animals; Antibodies, Neoplasm; Cell Line; Cell Transformation, Neoplastic; Epithelium; Female; Immunity, Cellular; Methylnitronitrosoguanidine; Mice; Mice, Inbred BALB C; Neoplasm Transplantation; Neoplasms, Experimental; Rats; Rats, Inbred F344; Tracheal Neoplasms; Transplantation Immunology; Transplantation, Isogeneic

The standard method of the C3H/10T1/2 cell transformation assay cannot adequately detect alkylating agents. A modification of the standard procedure as described by Bertram and Heidelberger using a large number of synchronized cells and high levels of toxicity was evaluated for transformation using several alkylating agents. By using this method, N-methyl-N' -nitro-N-nitrosoguanidine (MNNG), beta-propiolactone (BPL), methyl methanesulfonate (MMS), methylnitrosourea (MNU) and 1,3-propane sultone (PS) transformed these cells. However, methyl iodide (MI) failed to induce any transformed foci.

Topics: Alkylating Agents; Animals; Cell Cycle; Cell Line; Cell Transformation, Neoplastic; Drug Evaluation, Preclinical; Hydrocarbons, Iodinated; Methyl Methanesulfonate; Methylnitronitrosoguanidine; Methylnitrosourea; Mice; Propiolactone; Thiophenes

Adenomatosis of the colon and rectum (ACR) is an inherited form of cancer. Assuming that phenotypic expressions that appear in cell strains reflect its biological abnormalities, the study of cultured skin fibroblasts derived from individuals with an inherited form of cancer such as ACR provides a unique study for analysis of the oncogenic process. Growth disorders and increased susceptibility to tumor promoters and to transformation by an oncogenic RNA tumor virus have been demonstrated in these skin fibroblasts. We found that human skin fibroblasts (PF) derived from ACR individuals were sensitive to a chemical carcinogen. Cells treated only with various levels of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) underwent morphological alteration. The morphologically altered cells formed large cell aggregates when suspended in liquid growth medium above an agar base and grew to high saturation densities but did not form colonies in soft agar. Transformed cells were resistant to rechallenge of MNNG (1 microgram/ml) and showed prolonged life span compared to those untreated cells. However, no tumors were produced when cells were inoculated subcutaneously into nude mice. Data suggest that neoplastic transformation of these skin cells by chemical carcinogens is a multi-phase process.

Topics: Cell Transformation, Neoplastic; Cells, Cultured; Fibroblasts; Gardner Syndrome; Humans; Methylnitronitrosoguanidine; Skin; Tetradecanoylphorbol Acetate

Malignant transformation and mutation to ouabain-resistance is induced in cloned M2 mouse fibroblasts in vitro by treatment with N-methyl-N'-nitro-N-nitrosoguanidine and two derivatives of polycyclic aromatic hydrocarbons, the 3,4-diol derived from 7-methylbenz [a]anthracene and the 7,8-diol-9,10-oxide derived from benzo[a]pyrene. Transformation is an event more frequent than mutation, the ratio of transformation to mutation frequencies varied between 24 and 1118. Another hydrocarbon derivative, the 9,10-diol-7,8-oxide derived from benzo[a]pyrene, did induce ouabain-resistance without causing transformation. Induced ouabain-resistant clones did not behave like transformants, and transformants did not exhibit ouabain resistance. The significance of these findings is discussed.

Topics: Animals; Benz(a)Anthracenes; Benzopyrenes; Cell Transformation, Neoplastic; Cells, Cultured; Drug Resistance; Fibroblasts; Male; Methylnitronitrosoguanidine; Mice; Mice, Inbred C3H; Mutagens; Mutation; Mutation Rate; Ouabain

Assays that quantitate leukocyte transformation were used to determine the effect of two chemical and two physical mutagens on Epstein-Barr virus (EBV)-induced transformation of human umbilical cord leukocytes (HUCL). After treatment of HUCL with methyl methanesulfonate (MMS). UV light, or X-rays, lymphocyte transformation by EBV displayed a typical "survival" curve usually associated with agent toxicity in dividing cell populations. Viral transformation showed increasing resistance to each of these agents if the lymphocytes were treated after virus infection. No enhancement of viral transformation was detected following MMS, X-ray, or UV treatment. In contrast, after sublethal doses of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were administered, HUCL were more readily transformed by exogenous EBV. Also, in direct contrast to the other mutagens tested, EBV-induced transformation became markedly more sensitive to MNNG when leukocytes were treated after virus infection. Neither chemicals nor radiation alone was capable of initiating HUCL transformation, nor could infectious EBV be routinely recovered from transformed HUCL treated with MNNG or radiation. These results suggest that MNNG enhanced transformation by predisposing leukocytes toward transformation during EBV infection.

Topics: Cell Transformation, Neoplastic; Cells, Cultured; Herpesvirus 4, Human; Humans; Leukocytes; Methyl Methanesulfonate; Methylnitronitrosoguanidine; Mutagens; Ultraviolet Rays; X-Rays

C57L mouse xenotropic type C virus infection inhibited N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced in vitro transformation of embryo cells from inbred beagles. Treatment with MNNG induced in vitro neoplastic transformation in uninfected canine cells but not in C57L virus-infected canine cells; the C57L virus-infected canine cells not treated with MNNG also remained untransformed. In this dog cell system, preinfection with a C57L mouse xenotropic type C virus inhibited in vitro neoplastic transformation induced by MNNG.

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Dogs; Embryo, Mammalian; Methylnitronitrosoguanidine; Mice; Mice, Inbred C57BL; Mice, Nude; Neoplasm Transplantation; Neoplasms, Experimental; Retroviridae; Species Specificity; Transplantation, Heterologous

Topics: Animals; Cell Adhesion; Cell Transformation, Neoplastic; Cell Transformation, Viral; Graft Rejection; Methylnitronitrosoguanidine; Mice; Mice, Nude; Neoplasm Metastasis; Neoplasms, Experimental; Simian virus 40

The relationship between ageing and transformation has been investigated by a serial study of the changes in cell-surface morphology as normal and carcinogen-treated cells progressed in culture. A progressive increase in the density of cell surface microvilli occurred in association with the adoption of a more rounded profile and concomitant increase in the rate of cell detachment. These changes occurred earlier after carcinogen treatment, which appeared to indicate a carcinogen-induced acceleration of ageing. The alterations have also been described as characteristic of the transformed state. The observations suggest that the expression of in vitro transformation may be the result of continuous selection from a population with genetic instability and variable morphology.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Benzopyrenes; Carcinogens; Cell Membrane; Cell Survival; Cell Transformation, Neoplastic; Cells, Cultured; Cricetinae; Cricetulus; Lung; Methylnitronitrosoguanidine; Microscopy, Electron, Scanning; Time Factors

Exposure of C3H 10T1/2 Cl 8 cells, synchronized by release from confluence-induced arest of proliferation, to different concentrations of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) for 30 min at various points during the cell cycle causes dose-dependent toxicity (decrease in relative colony-forming efficiency or "survival") that increases linearly during the first G1 phase, reaches a maximum in early to middle S phase, and decreases during late S. In the course of the second S phase, toxicity again becomes maximal. The transformation rate (type III foci) increases and decreases with a similar pattern, increasing during the first G1 phase to a maximum during early S phase, subsequently decreasing, and then increasing again during the second S phase. Although periods of maximal toxicity and transformation roughly coincide with some portion of the S phase, the mechanisms underlying these phenomena appear to differ for the following reasons: (a) toxicity is linearly related to dose of MNNG, whereas the latter is linearly related to the logarithm of transformation rate, and (b) the ratio between toxicity and transformation varies with the cycle phase and the dose of MNNG.

Topics: Animals; Cell Cycle; Cell Division; Cell Line; Cell Survival; Cell Transformation, Neoplastic; DNA Replication; Methylnitronitrosoguanidine; Mice

The morphologic transformation induced in Syrian hamster embryo cells by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (0.25 microgram/ml of medium) is inhibited by posttreatment with antipain (6-600 microgram/ml), a protease inhibitor, but is unaffected by pretreatment. DNA replication relative to untreated controls is not affected by MNNG, antipain, or the combination of the two; no synergistic lethality of antipain and MNNG occurred as reflected in the cloning efficiency. Antipain was ineffective in influencing MNNG-induced sister chromatid exchanges, but it increased frequencies of chromosomal aberration (per metaphase) at 10, 26, and 40 hr when cells were treated with MNNG at 0.25 microgram/ml of medium followed by antipain 10 min later, the procedure used in the transformation studies. Antipain also increased the average number of aberrations at the second mitosis (34 hr) when the MNNG concentration was doubled. Chromatid exchanges increased 26 hr posttreatment with the combination of MNNG and antipain used for transformation. No difference in MNNG-induced aberrations was observed when antipain preceded MNNG by 24 hr. Although the mode of actin of antipain is unknown, antipain does not inhibit transformation by suppressing chromosomal rearrangements that could convert recessive mutations to the homozygous state.

Topics: Animals; Antipain; Cell Transformation, Neoplastic; Chromosome Aberrations; Cricetinae; DNA Replication; Metaphase; Methylnitronitrosoguanidine; Oligopeptides; Sister Chromatid Exchange

12-O-Tetradecanoylphorbol 13-acetate (TPA), a known tumor promoter, enhances the morphologic transformation of Syrian hamster embryo cells induced by low transforming concentrations of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (0.025-0.1 microgram/ml) without potentiation of cell lethality or of changes in sister chromatid exchanges (SCEs) or chromosomal aberration frequencies. When MNNG was added to logarithmically growing cultures and TPA was added 2, 24, or 48 hr later, no changes in SCE frequency relative to MNNG alone occurred. Similar results were obtained with TPA in cells that had been exposed to MNNG in stationary growth phase. Whereas no transformation occurred with TPA alone and pretreatment with TPA did not affect MNNG transformation, its addition 2 hr after MNNG reduced transformation frequency but addition 24-72 hr after MNNG increased the transformation frequency up to 6-fold. TPA had a minimal effect on increasing the transformation frequency (2-fold) induced by MNNG at 0.25 micrograms/ml, a high concentration. Of three polycyclic hydrocarbons, perylene, benzo[g,h,i]perylene, and benz[a]anthracene, known as weak or noncarcinogens, only benz[a]anthracene induced a very low transformation frequency; however, after TPA, transformation occurred with all three. Because the number of cells whose transformation was initiated by low doses of carcinogen is much larger than the number of cells giving rise to transformed colonies in the absence of TPA, the frequency of the initial event is greater than can be expected from point mutations. Furthermore, the promotional aspect of transformation is not accompanied by a parallel increase in SCE.

Topics: Animals; Cell Transformation, Neoplastic; Cells, Cultured; Chromosome Aberrations; Cocarcinogenesis; Cricetinae; Mesocricetus; Methylnitronitrosoguanidine; Phorbol Esters; Phorbols; Sister Chromatid Exchange

Foreskin-derived low-passage human cell populations were reproducibly transformed with chemical carcinogens when the cells were blocked in G1, released from the block, and treated with either the carcinogen N-methyl-N-nitro-N-nitrosoguanidine (MNNG) or with Aflatoxin B1 in the S period of the cell cycle. Arginine- and glutamine-deficient medium was required to effectively block the cells in the G1 period. Estradiol, insulin, anthralin or phorbol myristate acetate sensitized the cell population to carcinogen treatment when added 10 h before the carcinogen in early S period. Presensitized cells kept blocked in G1 period for 48 h or longer, released and treated in S period with MNNG or Aflatoxin B1 were not transformed; nor did transformation occur in presensitized cell populations treated in G2 (4.5 h), M (1.5 h) or G1 (8.2 h). Cells derived from carcinogen-treated presensitized cells grew as colonies in soft agar at 16-20 PDL. When cells derived from colonies isolated from the soft agar were injected subcutaneously into nude mice, tumors developed.

Topics: Aflatoxins; Animals; Cell Line; Cell Transformation, Neoplastic; Cells, Cultured; Culture Media; Humans; Interphase; Methylnitronitrosoguanidine; Mice; Mice, Nude; Neoplasms, Experimental; Skin

Studies of tumor induction on mouse skin have provided insight into the basis biology of chemical carcinogenesis, but molecular mechanisms have been more difficult to elucidate. Mouse epidermal cell cultures have proven to be a valuable model for performing mechanistic studies. Previous data have indicated that such cultures proliferate and differentiate in a manner highly analogous to epidermis in vivo. In addition, carcinogen metabolism, DNA repair, and responses to tumor promoters are quite similar in mouse skin in vivo and in vitro. Recent data have extended these observations toward defining the biological characteristics of initiated cells and elucidating the mechanism of action of promoters and antipromoters. When mouse epidermis is cultured under conditions of low extracellular Ca++, proliferation is enhanced and terminal differentiation is inhibited. Addition of Ca++ induces terminal differentiation. If cells are treated with carcinogens under low Ca++ conditions and subsequently switched to standard Ca++, cell colonies which do not terminally differentiate evolve. Such colonies continue to synthesize keratin, are subculturable, and may represent preneoplastic cells. In other experiments, epidermal cells derived from mouse skin treated with carcinogens in vivo also demonstrate prolonged in vitro survival and subculturability while controls have a limited lifespan. Such studies suggest that biological alterations can be detected in epidermal cells exposed to carcinogens well before and the phenotypic expression of neoplasia. Exposure of epidermal cells to phorbol-ester tumor promoters induces ornithine decarboxylase (ODC). This induction is enhanced by corticosteroids and markedly inhibited by retinoids. Ultraviolet light also induces ODC in epidermal cells, but kinetic studies suggest that the early pathway of induction (afferent to the nucleus) is different from that of phorbol esters. The later pathways (efferent from the nucleus-i.e., transcription and translation) appear to be similar. Retinoids have only a minor suppressive effect on ODC induction by UV while corticosteroids enhance UV induction to the same extent as seen with phorbol esters These results suggest that the site of retinoids is in the afferent pathway while steroids act on the efferent pathway.

Topics: Animals; Carcinogens; Cell Differentiation; Cell Transformation, Neoplastic; Cells, Cultured; Environmental Exposure; Enzyme Induction; Epidermal Cells; Epidermis; Methylnitronitrosoguanidine; Mice; Neoplasms, Experimental; Ornithine Decarboxylase; Skin Neoplasms; Tetradecanoylphorbol Acetate; Tretinoin

Chemical transformation of cultured human skin fibroblasts (PF) derived from individuals with hereditary adenomatosis of the colon and rectum is reported. Cells treated only with various levels of N-methyl-N'-nitro N-nitrosoguanidine (MNNG) underwent morphological alteration. The morphologically altered cells formed large aggregates when suspended in liquid growth medium above an agar base and grew to high saturation densities. One altered (MNNG, 1.0 microgram/ml) cell culture formed colonies in soft agar. Transformed cells were resistant to rechallenge of MNNG (l microgram/ml) and showed a more prolonged life-span compared to the untreated cells. Altered cells became heteroploid cells. However, no progressively growing tumors were produced when cells were inoculated subcutaneously into nude mice. The data suggest that chemical carcinogens alone may not induce neoplastic transformation of fibroblasts from humans genetically predisposed to cancer and that neoplastic transformation of these skin cells by chemical carcinogens might require the presence of a tumor promotor and the use of an immuno-privileged site in the nude mouse system.

Topics: Adenoma; Cell Transformation, Neoplastic; Colonic Neoplasms; Fibroblasts; Humans; Methylnitronitrosoguanidine; Rectal Neoplasms; Skin

The enhancement of chemical carcinogenesis was demonstrated in vitro in rat tracheal epithelium exposed in organ culture to N-methyl-N'-nitro- N-nitrosoguanidine (MNNG) followed by multiple exposures to 12-O-tetradecanoyl-phorbol-13-acetate (TPA). The explants were exposed to 0 or 0.0001 mu g MNNG per ml for 6 h on days 3 and 6 of culture, and then to 0 or 1.0 mu g TPA per ml for I h on days 9, 15, 21, and 27 of culture. Primary cell cultures were then established from epithelial outgrowths of these explants. Morphologically altered cells were seen in the primary cell cultures derived from MNNG + TPA-exposed explants on an average of 130 days after MNNG exposure. In primary cultures derived from explants exposed to either TPA or MNNG alone, morphologically altered cells were seen at 190 days and 210 days, respectively. No morphologically altered cells were seen in solvent control primary cell cultures. Only cultures containing morphologically altered cells could be repeatedly subcultured. Cell lines established from these cultures were inoculated i.m. into immunosuppressed isogeneic hosts at 150, 200, 250, 310, and 365 days following carcinogen exposure. All five explants exposed to both MNNG and TPA produced at least one cell line which formed carcinomas when inoculated 365 days following exposure, while only two of the five explants produced tumorigenic cell lines in the MNNG-only group. Cell lines from explants exposed to both MNNG and TPA were tumorigenic an average of 90 days earlier than cell lines from explants exposed to MNNG alone. None of the cell lines obtained from the explants exposed only to TPA were tumorigenic. These studies show that, following carcinogen exposure, TPA can enhance the oncogenesis of cultured respiratory tract epithelium in terms of increasing the frequency of transformation and decreasing the time at which transformation could first be observed.

Topics: Animals; Cell Transformation, Neoplastic; Cells, Cultured; Epithelium; Female; Methylnitronitrosoguanidine; Neoplasms, Experimental; Phorbols; Rats; Tetradecanoylphorbol Acetate; Time Factors; Trachea; Tracheal Neoplasms

Postconfluent cultures of Syrian hamster embryo cell strains were exposed to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or to combinations of MNNG, hydroxyurea (HU), caffeine (CF), and/or theophylline. At various intervals, [3H]thymidine incorporation into DNA was determined by autoradiography and liquid scintillation spectrometry. In parallel cultures, drug- and/or carcinogen-elicited cytotoxicity and carcinogen-induced morphological transformation were determined. The results indicate that: (a) MNNG induces, in a relatively small proportion of cells, a round of HU- and CF-suppressible [3H]thymidine incorporation superimposed on, or immediately followed by, a wave of inhibitor-resistant incorporation; (b) the transformation frequency varies with the detectable [3H]thymidine in the acid-insoluble fractions and the relative size of cell subpopulations undergoing HU- and CF-suppressible DNA replication; (c) the cells exposed to 1 mM CF or theophylline during the treatment with MNNG (0.5 microgram/ml), as well as for the first 24 hr after being plated for focus formation, develop statistically significant fewer (p less than 0.001) transformed foci, without measurable cytotoxic effects; and (d) at the doses used, HU inhibited [3H]thymidine incorporation of carcinogen-stimulated cells. It is concluded that MNNG-stimulated [3H]thymidine incorporation into DNA that is suppressible by both HU and CF may be associated with the initiation event(s) of morphological transformation; however, the nature of suppression by HU and CF appears to be different.

Topics: Animals; Autoradiography; Caffeine; Carcinogens; Cell Survival; Cell Transformation, Neoplastic; Cells, Cultured; Cricetinae; DNA; Dose-Response Relationship, Drug; Embryo, Mammalian; Hydroxyurea; Mesocricetus; Methylnitronitrosoguanidine; Stimulation, Chemical; Theophylline; Thymidine

Neoplastic transformation of primary fibroblast culture derived from esophagus tissue of a 52-year old male esophageal cancer patient was induced by chemical carcinogen (N-methyl-N'-nitro-N-nitrosoguanidine, MNNG) treatment. The transformed cells showed the biological and morphological properties characteristic of malignant cells, such as loss of contact inhibition, unlimited growth in vitro, aneuploidy, agglutinability by concanavalin A, formation of microvilli on the cell surface, growth on solid agar medium and tumor (fibrosarcoma) formation after heterotransplantation into immunosuppressed newborn mice.

Topics: Aneuploidy; Animals; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Concanavalin A; Disease Susceptibility; Esophageal Neoplasms; Esophagus; Fibroblasts; Fibrosarcoma; Humans; Male; Methylnitronitrosoguanidine; Mice; Middle Aged; Neoplasm Transplantation

58 MCA Cl 16 is an oncogenic methylcholanthrene-transformed variant of the non-transformed mouse embryo fibroblast cell line, C3H/10T1/2 Cl 8. Using two different protocols, we have isolated six temperature-sensitive mutants from N-methyl-N'-nitro-N-nitrosoguanidine treated cultures of 58 MCA Cl 16. C3H/10T1/2 Cl 8, 58 MCA Cl 16 and the six mutant lines were characterized with respect to several properties associated with the transformed state: morphology, saturation density, anchorage independence, cell surface morphology and growth in medium containing 1% fetal calf serum. In general, C3H/10T1/2 cells behaved as non-transformed, whether grown at 33 degrees C or 39.5 degrees C. The transformed parental line and all six mutants behaved as transformed cells at 33 degrees C. At 39.5 degrees C, only the parental transformed line retained the transformed phenotype. Three of the mutants revert towards non-transformed behavior at 39.5 degrees C for all of the properties tested. The remaining mutants are temperature-sensitive for some, but not all, transformed characteristics. Thus, while the expression of these transformed properties is sometimes coupled, we have been able to dissociate the expression of traits such as saturation density, anchorage independence and transformed morphology from each other. These mutants should prove to be valuable tools in the study of the mechanisms which underly the expression of the chemically-induced transformed state.

Topics: Animals; Carcinogens; Cell Adhesion; Cell Division; Cell Line, Transformed; Cell Separation; Cell Transformation, Neoplastic; Fibroblasts; Methylcholanthrene; Methylnitronitrosoguanidine; Mice; Mice, Inbred C3H; Microscopy, Electron, Scanning; Mutagenesis; Mutagens; Phenotype; Selection, Genetic; Temperature

Basal epidermal cells can be selectively maintained as a monolayer in culture medium containing a low ionic calcium concentration of 0.01-0.10 mM. Cessation of proliferation, maturation and shedding of squamous sheets can be induced in this population by increasing the calcium concentration above 0.1 mM. Since alterations in the regulation of proliferation and differentiation are associated with epidermal carcinogenesis in vivo, it appeared reasonable that changes in the phenotypic response to calcium might follow exposure to carcinogens in vitro. Support for this hypothesis was provided by the observation that malignant epidermal cells continued to proliferate when switched from low to high calcium medium, and could thus be selected from a mixture of such cells and a large excess of normal cells which did not survive after induced differentiation. Normal primary epidermal cells were plated in low calcium medium, treated on day 3 with a chemical carcinogen, maintained for 3-9 weeks in low calcium (0.02 mM) and then switched to high calcium medium (1.4 mM). After an additional 4 weeks, surviving epithelial colonies were fixed, stained with rhodamine and counted. Treatment of cultures with 7,12-dimethylbenz[a]anthracene or N-methyl-N'-nitro-N-nitrosoguanidine yielded 4-10 fold more colonies than solvent controls. Colony number was proportional to carcinogen dose for both agents, and increased with time in low calcium prior to selection by calcium increase. Cells obtained from colonies in treated cultures demonstrated characteristic epidermal morphology and keratinization, and could be subcultured, but did not grow in agar or produce tumors in syngeneic hosts. This model system represents a quantitative assay for carcinogen altered epithelial cell differentiation and may select for an early property of preneoplastic epidermal cells.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Calcium; Carcinogens; Cell Adhesion; Cell Differentiation; Cell Division; Cell Line, Transformed; Cell Transformation, Neoplastic; Cells, Cultured; Coculture Techniques; Coloring Agents; Dose-Response Relationship, Drug; Epidermal Cells; Epidermis; Methylnitronitrosoguanidine; Mice; Mice, Inbred BALB C; Phenotype; Rhodamines; Staining and Labeling

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

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

A recent epidemiological survey in China showed that there is a regional distribution of esophageal cancer and a correlation between mortality for this cancer and environmental factors, especially the consumption of pickled vegetables. A series of experimental studies with pickled vegetable extract were done using different in vitro biological systems. The results showed that pickled vegetable extract induced 6-thioguanine-resistant mutants in V79 cells and increased sister chromatid exchanges in the same cells and in Syrian hamster embryo cells. Pickled vegetables extract induced transformed foci in Syrian hamster embryo cells and in 3-methylcholanthrene initiated C3H/10T1/2 cells. The mutagenic, transforming and promoting activities of pickled vegetable extract seen in vitro conform with in vivo results and provide evidence for the presence of a mutagen and/or a carcinogen in pickled vegetable extract. A possible role of pickled vegetables consumption in the etiology of esophageal cancer is discussed.

Topics: Animals; Cell Transformation, Neoplastic; Cells, Cultured; China; Cricetinae; Cricetulus; Drug Resistance; Esophageal Neoplasms; Fibroblasts; Food Preservation; Food Preservatives; Male; Mesocricetus; Methylnitronitrosoguanidine; Mice; Mice, Inbred C3H; Mutagenesis; Mutagenicity Tests; Rats; Rats, Wistar; Sister Chromatid Exchange; Spices; Thioguanine; Vegetables

Topics: 4-Nitroquinoline-1-oxide; Animals; Benzopyrenes; Cell Transformation, Neoplastic; Cells, Cultured; Clone Cells; Cricetinae; Embryo, Mammalian; Female; Fibrinolysis; Fibroblasts; Mesocricetus; Methylnitronitrosoguanidine; Phenotype

The protein synthesis inhibitor cycloheximide, at a concentration of 0.08 microgram per milliliter, induced flat morphology within 24 to 48 hours and low saturation density in human osteosarcoma cells transformed by Kirsten murine sarcoma virus (Ki-MSV) or N-methyl-N' nitro-N-nitrosoguanidine. Removal of the protein synthesis inhibitor caused both transformed cells to revert to the transformed phenotype. The demonstration of cell-surface antigens, cross-reacted with antiserums induced by extracts of both types of transformed human cells, was dependent on the presence or absence of cycloheximide in the culture medium. The results show that protein synthesis is required to maintain the transformed state in virally or chemically transformed human cells.

Topics: Antigens, Surface; Cell Division; Cell Survival; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cells, Cultured; Cycloheximide; Gammaretrovirus; Humans; Methylnitronitrosoguanidine; Neoplasm Proteins; Sarcoma Viruses, Murine

Chemical carcinogens induce mutations to ouabain resistance in the transformable mouse fibroblast cell line C3H/10T1/2 Cl 8. The mutant phenotype is stable and heritable in the absence of selective agent, and dose--response curves for mutant frequency were obtained with N-menthyl-N'-nitro-N-nitrosoguanidine, 3-methylcholanthrene, benzo[a]-pyrene, N-acetoxy-N-2-acetylaminofluorene, and the anti-7,8-diol-9,10-epoxide of benzo[a]pyrene. The ratio of the malignant transformation frequency to the mutation frequency was 12 for benzo[a]pyrene and 21 for N-acetoxy-N-2-acetylaminofluorene. The development of the mutational assay reported here allows the use of this permanent cell line for comparison of mutation and transfomration frequencies and as a screening system for xenobiotics that pose mutagenic or carcinogenic hazards to mammalian cells.

Topics: 2-Acetylaminofluorene; Animals; Carcinogens; Cell Transformation, Neoplastic; Cells, Cultured; Dose-Response Relationship, Drug; Drug Resistance; Methylnitronitrosoguanidine; Mice; Mice, Inbred C3H; Mutation; Ouabain; Time Factors

Topics: Animals; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Cricetinae; Cricetulus; Methylcholanthrene; Methylnitronitrosoguanidine; Mice; Mice, Inbred C3H; Mutation; Protease Inhibitors

Topics: Animals; Cell Division; Cell Line; Cell Transformation, Neoplastic; DNA; Isoleucine; Kinetics; Methylnitronitrosoguanidine; Mice

Topics: Adenocarcinoma; Animals; Carcinoma, Squamous Cell; Cell Line; Cell Transformation, Neoplastic; Epithelium; Methylnitronitrosoguanidine; Neoplasms, Experimental; Rats; Time Factors; Tracheal Neoplasms

A subclone of ts 14/MNU/2, derived from a heteroploid (epithelial) monkey kidney cell line of BSC-1 and transformed in vitro by methylnitrosourea, expressed the transformed phenotype only at the elevated temperature of 39.5(0)C. Compared to the growth characteristics at 33(0)C, the transformed property was exhibited in the (1) high efficiency of plating at low cell densities, (2) colony morphology, (3) growth in absence of serum and (4) alterations in the quality and the quantity of chromosomal proteins in presence of low concentrations of serum. Such experimental systems may provide an effective control for studying molecular mechanisms related to transformation and neoplasia.

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Clone Cells; Cricetinae; Hot Temperature; Kidney; Methylnitronitrosoguanidine; Models, Biological; Phenotype

Topics: Adenocarcinoma; Cell Membrane; Cell Transformation, Neoplastic; Cells, Cultured; Humans; Male; Methylnitronitrosoguanidine; Microscopy, Electron, Scanning; Prostate; Prostatic Neoplasms; Time Factors

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

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

Topics: Alkylating Agents; Cell Line; Cell Survival; Cell Transformation, Neoplastic; DNA Repair; Dose-Response Relationship, Drug; Ethyl Methanesulfonate; Methyl Methanesulfonate; Methylnitronitrosoguanidine; Mutagens

Topics: Adenosine Triphosphatases; Animals; Benzopyrenes; Cell Transformation, Neoplastic; Cells, Cultured; Cricetinae; Cytological Techniques; Genetic Techniques; Hypoxanthine Phosphoribosyltransferase; Methylnitronitrosoguanidine; Mutagens; Mutation

Somatic mutation and neoplastic transformation of diploid Syrian hamster embryo cells were examined concomitantly. Mutations induced by benzo[a]pyrene and N-methyl-N'-nitro-N-nitrosoguanidine were quantitated at the hypoxanthine phosphoribosyltransferase and Na(+)/K(+) ATPase loci and compared to phenotypic transformations measured by changes in cellular morphology and colony formation in agar. Both cellular transformations had characteristics distinct from the somatic mutations observed at the two loci. Morphological transformation was observed after a time comparable to that of somatic mutation but at a frequency that was 25- to 540-fold higher. Transformants capable of colony formation in agar were detected at a frequency of 10(-5)-10(-6), but not until 32-75 population doublings after carcinogen treatment. Although this frequency of transformation is comparable to that of somatic mutation, the detection time required is much longer than the optimal expression time of conventionally studied somatic mutations. Neoplastic transformation of hamster embryo cells has been described as a multistep, progressive process. Various phenotypic transformations of cells after carcinogen treatment may represent different stages in this progressive transformation. The results are discussed in this context and the role of mutagenesis in the transition between various stages is considered. Neoplastic transformation may be initiated by a mutational change, but it cannot be described completely by a single gene mutational event involving a dominant, codominant, or X-linked recessive locus. Neoplastic transformation induced by chemical carcinogens is more complex than a single gene mutational process. Thus, this comparative study does not give experimental support to predictions of the carcinogenic potential of chemicals based on a simple extrapolation of the results obtained from conventional somatic mutation assays.

Topics: Adenosine Triphosphatases; Benzopyrenes; Cell Adhesion; Cell Line; Cell Transformation, Neoplastic; Dose-Response Relationship, Drug; Hypoxanthine Phosphoribosyltransferase; Methylnitronitrosoguanidine; Mutation; Time Factors

Effects of Propionibacterium acnes on production of antibodies against tumor-specific membrane antigens were investigated in syngeneic inbred BD IV and BD VI rats. BD rat liver cell lines transformed in vitro by chemical carcinogens were used as target cells for tumor-specific antigens. By membrane immunofluorescence, antibodies against these rat liver cell lines were detected in syngeneic BD rat sera. Antibodies were produced in syngeneic rats under the adjuvant effect of heat-killed P. acnes only. In assays with various target cells and absorption experiments, the antibodies reacted with a tumor-specific individual antigen or tumor-specific cross-reacting antigen on the surfaces of transformed BD rat liver cells. No antibodies against these antigens were found in the sera obtained from syngeneic rats immunized with either the transformed cell lines or P. acnes but not with both. Freund's complete adjuvant did not induce antibodies against these tumor-specific antigens.

Topics: Animals; Antibodies, Neoplasm; Antibody Specificity; Cell Transformation, Neoplastic; Cells, Cultured; Dimethylnitrosamine; Liver Neoplasms; Methylnitronitrosoguanidine; Neoplasms, Experimental; Propionibacterium acnes; Rats

Cultured fibroblast cells derived from a skin biopsy sample taken from normal human adult were exposed to a potent carcinogen, 4-nitroquinoline 1-oxide. Alterations of cell growth pattern such as higher density and piling up of cells were noticed in some fractions of cultures that were successively subcultured after nitroquinoline oxide treatment. Morphologically altered cells retained this growth pattern and became established lines of transformed cells without showing the limited life-span characteristic of normal cells in culture. The transformed cells showed a higher saturation density and the ability to grow in soft agar, properties that are usually correlated with neoplastic transformation of cells in culture. Selection of preexisting transformed human cells as a mechanism of this observed transformation seemed unlikely because clones of these normal cells could also be used to assess the transforming effect of nitroquinoline oxide. Preliminary results suggest that numerous cell divisions were required for the development of the transformation after nitroquinoline oxide treatment of these human cells. When the transformed cell lines were injected subcutaneously into nude (athymic) mice, solid tumors were produced at the site of inoculation. Treatment with N-methyl-N'-nitro-N-nitrosoguanidine also induced cell transformation, in a manner similar to treatment with nitroquinoline oxide. However, transformation was not induced with (i) 4-aminoquinoline 1-oxide (a noncarcinogenic derivative of 4-nitroquinoline 1-oxide), (ii) 3-methylcholanthrene (a carcinogen that cannot be metabolically activated by the target cells employed), or (iii) the solvent dimethyl sulfoxide.

Topics: 4-Nitroquinoline-1-oxide; Animals; Carcinogens; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Contact Inhibition; Humans; Methylnitronitrosoguanidine; Mice; Neoplasm Transplantation; Neoplasms, Experimental

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

Topics: Animals; Animals, Newborn; Cell Line; Cell Transformation, Neoplastic; Epoxy Compounds; In Vitro Techniques; Intercellular Junctions; Methylcholanthrene; Methylnitronitrosoguanidine; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; Neoplasms, Experimental; Skin; Transplantation, Homologous; Transplantation, Isogeneic

The present study was performed in order to determine whether type III transformed foci induced by N-methyl-N'-nitro-N-nitrosoguanidine originate from the small subpopulation of cells stimulated by the carcinogen to enter DNA synthesis. During the last 30 min of variable treatment periods using different doses of N-methyl-N'-nitro-N-nitrosoguanidine, administered alone or in association with the thymidine analogue, 5'-bromo-2'-deoxyuridine (0.98 x 10(-5)M), the density-inhibited monolayers of hamster embryo cells were exposed to fluorescent light and then assayed for abnormal growth patterns by the focus formation method. Mock-irradiated cultures as well as monolayers whose medium lacked N-methyl-N'-nitro-N-nitro-soguanidine, 5'-bromo-2'-deoxyuridine, or both, served as controls. The cytotoxicity of 5'-bromo-2'- deoxyuridine + N-methyl-N'-nitro-N-nitrosoguanidine + photolysis (BMP) protocol on confluent as well as logarithmically growing hamster embryo cells was estimated in single-cell survival experiments. Plating efficiency determinations have demonstrated that, unlike their actively growing counterparts, confluent hamster embryo cell monolayers are extremely resistant to the cytotoxic effects of the BMP protocol. The quantitative transformation assays indicated that: (1) in non-illuminated cultures addition of 5'-bromo-2'-deoxyuridine to carcinogen-containing medium does affect transformation frequency of hamster embryo cells in the sense that the incidence of type III foci did not subside at later intervals during the post-carcinogen administration period as it did in the absence of the analogue; (2) irradiation of N-methyl-N'-nitro-N-nitrosoguanidine and halogenated pyrimidine analogue-treated cultures with fluorescent light practically suppressed transformation; (3) analogue-added and analogue-removed experiments pointed out that the event(s) on which 5'-bromo-2'-deoxyuridine fluorescent light sensitization of morphological transformation largely depends, takes place between 5 and 15 h after N-methyl-N'-nitro-N-nitrosoguanidine administration, i.e., during the period of maximal carcinogen-stimulated DNA synthesis; and (4) neither fluorescent light nor 5'-bromo-2'-deoxyuridine, singly or in combination, were able to transform cultures of hamster embryo cells. These findings are strong indirect arguments for the concept that carcinogen-induced DNA synthesis and the initiation of transformed clones are causally related.

Topics: Animals; Bromodeoxyuridine; Cell Division; Cell Survival; Cell Transformation, Neoplastic; Cells, Cultured; Cricetinae; DNA; Methylnitronitrosoguanidine; Photolysis; Time Factors

A cell line derived from a normal beagle embryo was treated in vitro with various levels of N-methyl-N'-nitro-N-nitrosoguanidine or dimethyl sulfoxide (control). Cells treated only with the carcinogen underwent morphologic alteration in vitro, and one of these altered cell lines produced tumors subcutaneously when injected into NIH nude mice. The tumorigenic transformed line formed larger cell aggregates and grew in this aggregate form when suspended in liquid growth medium above an agar base.

Topics: Animals; Carcinogens; Cell Aggregation; Cell Line; Cell Transformation, Neoplastic; Cells, Cultured; Culture Media; Dogs; Embryo, Mammalian; Methylnitronitrosoguanidine; Mice; Mice, Nude; Neoplasm Transplantation; Neoplasms, Experimental; Sulfoxides; Transplantation, Heterologous

A number of chemical and physical agents were screened to determine their effectiveness in inducing simian virus (SV40) production in a virogenic clone of SV40-transformed Chinese hamster cells. Mitomycin C (MC) was the most effective inducing agent, and MC induction was further characterized. It was found that levels of infectious SV40 DNA were increased above control levels as early as 6 h after addition of MC to the culture medium and reached maximum levels by 48 h. Virus capsid (V) antigen and virions followed with a lag of about 24 h. V antigen production was sensitive to hydroxyurea, suggesting a dependence on virus DNA synthesis. The proportion of virus-producing cells (infectious centres) and the virus burst per cell were both stimulated by MC. Studies of 3H-thmidine incorporation demonstrated that the rate of SV40 DNA synthesis was maximal at 48 h post-induction, at which time cellular DNA synthesis was almost abolished. Caffeine, at doses not toxic to non-induced cells, strongly inhibited SV40production in both non-induced and induced cells, suggesting some role for DNA repair mechanisms.

Topics: Animals; Bucladesine; Caffeine; Cell Line; Cell Transformation, Neoplastic; Clone Cells; Cricetinae; Cycloheximide; DNA, Neoplasm; DNA, Viral; Hydroxyurea; Idoxuridine; Methylnitronitrosoguanidine; Mitomycins; Simian virus 40; Virus Replication

Five temperature-sensitive mutants (ts I to 5) were isolated from a stock of the Moloney strain of murine sarcoma leukaemia virus complex which had been mutagenized by ultraviolet irradiation or N-methyl-N'-nitro-N-nitrosoguanidine. In mouse cells at the non-permissive temperature the mutants formed fewer foci of transformed cells than at the permissive temperature. The ts mutants were characterized by testing: (I) murine leukaemia virus (MuLV) clones from the ts complex, (2) the effect of additional wild type MuLV on focus formation, (3) focus formation in rat cells and (4) focus formation with pseudotypes rescued from non-producer cells. Two mutants (ts 1 and ts 3) were found to be ts MuLVs which did not possess heat labile virion proteins and were not ts in post-penetration helper functions necessary for the fixation of sarcoma virus transformation. The remaining three mutants (ts 2, ts 4 and ts 5) were ts murine sarcoma viruses which, however, showed no temperature-sensitive effect on the maintenance of transformed cell morphology nor on colony forming efficiency in soft agar.

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Gammaretrovirus; Leukemia Virus, Murine; Methylnitronitrosoguanidine; Mutagens; Mutation; Rats; Sarcoma Viruses, Murine; Temperature; Ultraviolet Rays; Viral Proteins; Virus Replication

Human osteosarcoma (HOS) clonal cells transformed in vitro by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were characterized, and compared to non-producer HOS cells transformed by Kirsten murine sarcoma virus (Ki-MSV). The MNNG- and virus-transformed cells grew in the aggregate form above an agar base, grew in soft agar, and had a high fibrinolytic activity. When inoculated into nude mice, all the chemically or virally altered cells produced tumors or tumor nodules. When transplanted into ATS-treated hamsters, the cells transformed by MNNG (0.01 mug/ml) and Ki-MSV produced tumors but MNNG (0.1 mug/ml) transformed cells did not produce tumors. The control HOS cells did not grow in the aggregate form but formed colonies in soft agar, and had low fibrinolytic activity and no capacity to form tumors in nude mice and ATS-treated hamsters. However, one of the control clonal lines had a high level of fibrinolytic activity. Cellular aggregation properties of human transformed cells did appear to correlate with tumorigenicity in nude mice.

Topics: Animals; Cell Aggregation; Cell Line; Cell Transformation, Neoplastic; Clone Cells; Culture Media; Humans; Methylnitronitrosoguanidine; Osteosarcoma; Sarcoma, Experimental

The time of susceptibility of cells to lymphotoxin during carcinogenesis was determined. At different stages of in vitro chemical carcinogen-induced neoplastic transformation, colony formation of guinea pig cells was evaluated with lymphotoxin obtained from syngeneic nonimmune leukocytes. Cells exhibiting sequential morphological alteration, morphological transformation, and neoplastic transformation had been preserved in liquid nitrogen and, after reintroduction in culture, were analyzed simultaneously for their susceptibility to lymphotoxin. Morphologically altered and morphologically transformed cells at subcultures prior to neoplastic transformation were resistant to lymphotoxin inhibition of colony formation. Cells neoplastically transformed by N-methyl-N'-nitro-N-nitrosoguanidine in vitro or by N-methyl-N'-nitro-N-nitrosoguanidine or diethylnitrosamine with the host-mediated, in vivo-in vitro method were susceptible and exhibited a quantitatively culture-specific degree of colony inhibition. The parental noncloned and cloned neoplastically transformed cells in each series, furthermore, exhibited similar degrees of colony inhibition, which indicates that lymphotoxin susceptibility developed concomitant with, or close to, the time of neoplastic transformation and remained stable during subsequent cell generations.

Topics: Animals; Ascitic Fluid; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Clone Cells; Diethylnitrosamine; Guinea Pigs; Leukocytes; Lymphotoxin-alpha; Male; Methylnitronitrosoguanidine; Neoplasms, Experimental; Time Factors

Topics: 4-Nitroquinoline-1-oxide; Acetoxyacetylaminofluorene; Animals; Carcinogens; Cell Line; Cell Survival; Cell Transformation, Neoplastic; Cricetinae; Dose-Response Relationship, Drug; Dose-Response Relationship, Radiation; Male; Mesocricetus; Methylnitronitrosoguanidine; Neoplasm Transplantation; Transplantation, Homologous; Ultraviolet Rays

Three cell lines were established from embryos of DDD, AKR, and C3H mouse strains based on "3T3" transfer schedule. These cell lines were designated as Y-DD, Y-AK, and Y-CH, Y-DD and Y-CH showed a gradually increasing growth rate during the course of their establishment, whereas Y-ak indicated a constant growth rate, a low level of saturation density, and a tetraploid range of chromosome mode. In transformation experiment, control cultures of Y-DD and Y-CH already lost a density-dependent inhibition, but Y-AK was found to be very sensitive to density-dependent inhibition. Y-AK showed a higher activity in benzo[a]pyrene metabolism (4100 pmol/10(6) cells/24 hr) than Y-DD and Y-CH (700 and 2600 pmol/10(6) cells/24 hr).

Topics: 4-Nitroquinoline-1-oxide; 9,10-Dimethyl-1,2-benzanthracene; Animals; Benzopyrenes; Cell Division; Cell Line; Cell Transformation, Neoplastic; Contact Inhibition; Karyotyping; Methylnitronitrosoguanidine; Mice; Mice, Inbred AKR; Mice, Inbred C3H; Mice, Inbred Strains; Species Specificity

Hamster embryonic fibroblasts were treated directly with various concentrations of methylnitrosocyanamide (MNC), a nitrosated product of methylguanidine (MG) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Then they were examined for chromosomal aberrations, morphological transformation and mutations resistant to 8-azaguanine (8AG) and 6-thioguanine (6TG). Direct treatment with 2 to 10 X 10(-6) M MNC caused a marked, dose-dependent appearance of 8AG- and 6TG-resistant mutations. The ability of MNC to induce mutations was similar to that of MNNG. Cultured embryonic fibroblasts in metaphase plates also showed a marked dose-dependent increase in chromosomal aberrations within 24 h after direct treatment with MNC or MNNG. Moreover, MNC and MNNG caused similar rates of morphological transformation.

Topics: Animals; Azaguanine; Cell Line; Cell Transformation, Neoplastic; Chromosome Aberrations; Chromosomes; Cricetinae; Dose-Response Relationship, Drug; Drug Resistance; Guanidines; Mesocricetus; Methylguanidine; Methylnitronitrosoguanidine; Mutation; Nitrosamines; Thioguanine

This study was undertaken to determine the effect of ulcer induced by iodoacetamide on the development of gastric carcinoma by N-methyl-N'-nitro-N-nitrosoguanidine in male Wistar rats. Fifty-six of the 62 ulcers induced by IAM were located in the fundic gland area along the limiting ridge. The incidence of fundic carcinoma was 16% in the groups treated with IAM and MNNG, while no fundic carcinoma was found in the group treated with MNNG alone. This difference was statistically significant. All the carcinomas in the fundic gland area were confined within the ulcer itself or its scar tissue, produced by IAM. These findings indicate that if an ulcer is present, carcinoma develops even in the fundic mucosa which is, if intact, resistant to the carcinogenic stimulation of MNNG. It was concluded that gastric ulcer predisposes the development of gastric carcinoma.

Topics: Adenocarcinoma; Adenoma; Animals; Cell Transformation, Neoplastic; Drug Synergism; Iodoacetamide; Iodoacetates; Male; Methylnitronitrosoguanidine; Neoplasms, Experimental; Rats; Stomach; Stomach Neoplasms; Stomach Ulcer

Topics: Cell Adhesion; Cell Transformation, Neoplastic; Cells, Cultured; Clone Cells; Culture Media; Diploidy; Fibroblasts; Humans; Methylnitronitrosoguanidine; Mutation

Using an organ culture/cell culture system, we transformed rat tracheal epithelial cells in vitro by exposure to MNNG. Ten tracheal organ cultures per group were exposed twice (at days 3 and 6) to 0,0.001, 1.0 or 10.0 microgram MNNG/ml of medium. Following this exposure, the explants were placed on the bottom of culture dishes to initiate epithelial cell outgrowths and establish primary cultures. Each explant was replanted 8-10 times to produce multiple outgrowths. The number of primary cultures and the number of subsequently established cell lines obtained was carcinogen-dose-dependent. The average number of primary epithelial cell cultures per explant after exposure to 0, 0.001 1.0 and 10.0 microgram MNNG/ml was 1.3, 1.5, 3.3, and 4.6, respectively. The average yield of cell lines per explant in these groups was 0, 0.8, 1.3, and 2.0, respectively. It is apparent that cell lines could only be established from carcinogen-exposed epithelium. These cell lines are currently being tested for tumorigenicity in vivo. To date, of 35 cell lines tested between the 5th and 15th passages, 5 cell lines from the 10 microgram MNNG/ml group and 2 from the 1.0 microgram MNNG/ml group have produced palpable tumors upon injection into immunosuppressed recipients.

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Epithelial Cells; Epithelium; Female; Methylnitronitrosoguanidine; Organ Culture Techniques; Rats; Trachea

Topics: Animals; Animals, Newborn; Carcinogens; Cell Count; Cell Transformation, Neoplastic; Cells, Cultured; Cricetinae; Culture Media; Drug Evaluation, Preclinical; Female; Male; Methylnitronitrosoguanidine; Neoplasm Transplantation; Neoplasms, Experimental; Transplantation, Homologous

Treatment of Syrian hamster embryo cells with diverse classes of chemical carcinogens enhanced transformation by a carcinogenic simian adenovirus, SA7. Optimal enhancement was a function of time of chemical addition in relation to time of virus addition and cell transfer. Aflatoxin B1 (AFB1) and the polycyclic hydorcarbons, benzo(a)pyrene (B(a)P), 3-methylcholanthrene (MCA), and 7,12-dimethylbenz(a)anthracene (DMBA) enhanced SA7 transformation when added prior to virus, but inhibited transformation when added after virus absorption and cell transfer. The enhancement of SA7 transformation was maximal when cytosine arabinoside, caffeine and 6-acetoxy-benzo(a)pyrene (6-ac-B(a)P) were added after virus, but minimal when added before virus. A third class of chemicals, including beta-propiolactone (beta-PL), methyl methanesulfonate (MMS), N-acetoxy-2-acetylaminofluorene (Ac-AAF), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and methylazoxymethanol acetate (MAM-ac), enhanced SA7 transformation added before, or after, virus inoculation and cell transfer. All chemicals which induced changes in DNA sedimentation in alkaline sucrose gradients and unscheduled DNA (repair) synthesis in hamster cells, increased the frequency of SA7 transformation. However, several chemicals such as dibenz(a,h)anthracene (DB(a,h)A), benzo(e)pyrene (B(e)P), cytosine arabinoside, and caffeine enhanced SA7 transformation but did not induce DNA sedimentation changes or repair. Chemicals that cause DNA damage, which can be repaired by hamster cells, may enhance viral transformation by providing additional sites for integration of viral DNA during the repair process. Chemicals that apparently do not induce DNA repair synthesis may enhance viral transformation by incorporation of viral DNA into gaps in cell DNA at sites of unrepaired damage during scheduled DNA synthesis.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Acetoxyacetylaminofluorene; Adenoviridae; Aflatoxins; Benzopyrenes; Caffeine; Carcinogens; Cell Line; Cell Transformation, Neoplastic; Cytarabine; DNA Repair; DNA Replication; Methyl Methanesulfonate; Methylcholanthrene; Methylnitronitrosoguanidine; Mutagens; Oncogenic Viruses; Propiolactone

Topics: Cell Line; Cell Transformation, Neoplastic; Fibroblasts; Humans; Methylnitronitrosoguanidine; Xeroderma Pigmentosum

Among several lines of cultured rat hepatocytes examined, one line, IAR-22, contained enzyme III as well as enzyme I of the isozymes of branched-chain amino acid transaminase (EC 2.6.1.42) and this cell line was tumorigenic. None of the cell lines tested contained enzyme II (EC 2.6.1.6) or tyrosine transaminase (EC 2.6.1.5) and addition of dexamethasone did not induce these transaminases. The PC-1 line, containing only enzyme I and having a high percentage of diploid cells, showed deviation of its chromosomal number and tumorigenicity, but no change in its isozyme pattern when transformed with N-methyl-N'-nitro-N-nitrosoguanidine. Therefore, judging from expression of isozymes of the transaminase, this transformed cell line retained moderate differentiation.

Topics: Amino Acids; Cell Line; Cell Transformation, Neoplastic; Chromosomes; Isoenzymes; Liver; Methylnitronitrosoguanidine; Time Factors; Transaminases

The chromosomes of five chemical carcinogen-transformed strain 2 guinea pig fetal cell lines were identified by G and C banding techniques and were compared with the normal karyotype from secondary untreated cultures. One line transformed by benzo(a)pyrene had a diploid constitution with no G and C band alterations. Three lines were near diploid, one was near tetraploid, and each contained abnormal chromosomes. A 3-methylcholanthrene line had an abnormal metacentric chromosome formed by centric fusion of two nonhomologous autosomes. The three N-methyl-N'-nitrosoguanidine or diethylnitrosamine cell lines exhibited submetacentric or subtelocentric abnormal chromosomes originating from translocations between two No. 1 or a No. 1 and another autosome. The involvement of Chromosome 1 may be due to its association with nucleolar organization. The greater frequency of contact between such chromosomes, compared to other autosomes, creates an increased risk of chromatid exchange possibly explaining their frequent participation in abnormal chromosome formation or nondisjunction.

Topics: Benzopyrenes; Carcinogens; Cell Transformation, Neoplastic; Cells, Cultured; Chromosome Aberrations; Chromosome Mapping; Chromosomes; Diethylnitrosamine; Methylcholanthrene; Methylnitronitrosoguanidine; Polyploidy

Full-thickness skin grafts from either BN (Ag-B3) or WF (Ag-B2) rats were transplanted to WF recipients of the same sex. Six to seven days after grafting, recipients were challenged with isografts of a chemically induced rat colon carcinoma, NG-W1. In three of four experiments, mean challenge tumor volumes were greater after allografting than after skin isografting. Tumor incidences, however, were no different in rats after skin allograft or isograft placement. When isografts of a polyoma virus-induced sarcoma, P-W13, were used to challenge WF rats after skin grafting, tumor incidence was significantly greater in animals which has received allografts, whether or not they also had been immunized to P-W13 before challenge. Thus, in otherwise untreated inbred rats, grafting of full-thickness skin from donors differing at a major histocompatibility locus led to facilitated growth of solid tumor isografts in animals undergoing allograft rejection.

Topics: Adenocarcinoma; Animals; Cell Transformation, Neoplastic; Female; Male; Methylnitronitrosoguanidine; Neoplasm Transplantation; Polyomavirus; Rats; Rats, Inbred BN; Rats, Inbred WF; Sarcoma, Experimental; Skin Transplantation; Time Factors; Transplantation, Homologous; Transplantation, Isogeneic

Topics: Benzopyrenes; Carcinogens; Cell Transformation, Neoplastic; Estradiol; Humans; Methylnitronitrosoguanidine; Simian virus 40

Topics: Animals; Biological Assay; Cell Line; Cell Transformation, Neoplastic; Dose-Response Relationship, Drug; Humans; Karyotyping; Methylnitronitrosoguanidine; Mice; Mice, Nude; Nitrosoguanidines; Osteosarcoma; Time Factors

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

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

Epithelial-like cells originating from the livers of 10-day and 8-week-old BD rats were established in culture. The cells were treated in vitro for 1 or 4 weeks with dimethylnitrosamine (DMN) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Although some structural changes were observed in treated cells, it was not possible to score for morphological transformation in vitro. Newborn syngeneic rats were injected with 1.5-2 times 10(6) treated or 1.5-5 times 10(6) control cells at various times up to 38 weeks from the beginning of treatment with the carcinogen. Following the injection of DMN-treated cells, a total of 32 of the 42 injected rat developed tumours, of which 17 were epithelial, 10 carcinosarcomas and 5 fibrosarcomas. Following the injection of the MNNG-treated cells into 61 rats, a total of 30 tumours were observed, including 8 carcinomas, 9 carcinosarcomas and 13 fibroscarcomas. Tumours, mainly of the mesenchymal type, were also observed in rats inoculated with control cells but at a lower frequency. The observation observed in rats inoculated with control cells but at a lower frequency. The observation of mesenchymal tumours is attributed to the presence of a mixed population of epithelial and mesenchymal cells in the orginal culture.

Topics: Animals; Carcinosarcoma; Cell Line; Cell Transformation, Neoplastic; Cells, Cultured; Dimethylnitrosamine; Epithelial Cells; Epithelium; Fibrosarcoma; In Vitro Techniques; Liver; Mesenchymoma; Methylnitronitrosoguanidine; Neoplasm Metastasis; Neoplasm Transplantation; Neoplasms, Experimental; Nitrosamines; Nitrosoguanidines; Rats; Rats, Inbred Strains; Sarcoma, Experimental; Transplantation, Isogeneic

The introduction of a polycyclic hydrocarbon such as benzo(alpha)pyrene (BP) into normal golden hamster embryo cell cultures results, in addition to cytotoxicity, in malignant cell transformation. Studies on the effect of different doses of BP on the normal cells showed that the frequency of transformed colonies was directly related to the dose of the carcinogen. Analysis of this dose-response curve suggests a one-event ("one-hit") response for transformation by this carcinogen. The one-event response for transformation by carcinogenic polycyclic hydrocarbons and the fact that these carcinogens bind to DNA in susceptible cells suggests that transformation can involve a single alteration in the genetic constitution of the treated cells. Carcinogens may, therefore, produce somatic mutations, some of which may involve the genes that control malignancy. Recently, considerable progress has been made in developing models for the study of chemical mutagenesis in mammalian cells. Using resistance to 8-azaguanine as a marker, positive correlations between mutagenicity and transformation were obtained with chemically reactive carcinogens such as N-acetoxy-N-2-fluorenyl-acetamide, N-methyl-N'-nitro-N-nitrosoguanidine and K-region epoxides of polycyclic hydrocarbons. However, no such correlations were obtained with the carcinogenic polycyclic hydrocarbons themselves, since the cell lines used in chemical mutagenesis do not metabolize these carcinogens. In order to obtain better correlations, we have developed a cell-mediated mutagenic assay with carcinogenic hydrocarbons in which Chinese hamster cells, which are susceptible for mutagenesis, were co-cultivated with lethally irradiated rodent cells that can metabolize these compounds. Using this cell mediated assay, we obtained mutagenesis with the carcinogenic hydrocarbons 7,12-dimethylbenz(alpha)anthracene (DMBA), BP, 3-methylcholanthrene and 7-methylbenz(alpha)anthracene; the most potent carcinogen, DMBA, gave the highest frequency of mutations. The polycyclic hydrocarbons, pyrene and benz(alpha)anthracene, which are not carcinogenic were also not mutagenic. We have therefore demonstrated a relationship between the carcinogenecity of polycyclic hydrocarbons and their mutagenicity in mammalian cells, without having to isolate their reative metabolic intermediates. It should be possible to use in this system human cells from different organs and individuals to screen for environmental chemicals hazardous to humans whi

Topics: Acetoxyacetylaminofluorene; Animals; Bacterial Physiological Phenomena; Benzopyrenes; Carcinogens; Cell Transformation, Neoplastic; Cells, Cultured; Clone Cells; Cricetinae; Hydrocarbons; Methylnitronitrosoguanidine; Mutagens; Mutation; Virus Physiological Phenomena

The repair of single-strand breaks induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in mouse embryo fibroblast DNA was studied using an alkaline sucrose sedimentation procedure with cells synchronized by arginine deprivation. Parallel studies in synchronized cells examined the effects of MNNG on malignant transformation and cell death. Cells in late G1 were maximally transformed, while cells in S-phase were maximally killed; by contrast, the repair capacity in both cases was similarly rapid. Arginine-deprived cells showed a low level of DNA repair, but the extent of cell death and transformation was comparable to that in rapidly repairing cells in G1 and S, respectively. Thus, in this system there is no direct correlation among DNA repair, cell transformation, and lethality.

Topics: Animals; Cell Division; Cell Survival; Cell Transformation, Neoplastic; DNA; DNA Repair; Fibroblasts; Methylnitronitrosoguanidine; Mice; Molecular Weight; Nitrosoguanidines