methylnitronitrosoguanidine and Fibrosarcoma

Cadmium is a human carcinogen that affects cell proliferation, apoptosis and DNA repair processes that are all important to carcinogenesis. We previously demonstrated that cadmium inhibits DNA mismatch repair (MMR) in yeast cells and in human cell-free extracts (H.W. Jin, A.B. Clark, R.J.C. Slebos, H. Al-Refai, J.A. Taylor, T.A. Kunkel, M.A. Resnick, D.A. Gordenin, Cadmium is a mutagen that acts by inhibiting mismatch repair, Nat. Genet. 34 (3) (2003) 326-329), but cadmium also inhibits DNA excision repair. For this study, we selected a panel of three hypermutable tetranucleotide markers (MycL1, D7S1482 and DXS981) and studied their suitability as readout for the mutagenic effects of cadmium. We used a clonal derivative of the human fibrosarcoma cell line HT1080 to assess mutation levels in microsatellites after cadmium and/or N-methyl-N-nitro-N-nitrosoguanidine (MNNG) exposure to study effects of cadmium in the presence or absence of base damage. Mutations were measured in clonally expanded cells obtained by limiting dilution after exposure to zero dose, 0.5 microM cadmium, 5 nM MNNG or a combination of 0.5 microM cadmium and 5 nM MNNG. Exposure of HT1080-C1 to cadmium led to statistically significant increases in microsatellite mutations, either with or without concurrent exposure to MNNG. A majority of the observed mutant molecules involved 4-nucleotide shifts consistent with DNA slippage mutations that are normally repaired by MMR. These results provide evidence for the mutagenic effects of low, environmentally relevant levels of cadmium in intact human cells and suggest that inhibition of DNA repair is involved.

Topics: Base Pair Mismatch; Cadmium; DNA Repair; Fibrosarcoma; Humans; Methylnitronitrosoguanidine; Microsatellite Repeats; Mutagens; Mutation; Tumor Cells, Cultured

Current experimental models of pancreatic cancer either fail to reproduce the ductal phenotype or cause simultaneous cancers in other organs also. To develop an animal of pancreatic cancer that accurately mimics the human condition, we restricted carcinogenic exposure to the pancreas and specifically targeted ductal epithelial cells. Three different carcinogens were either implanted directly into the pancreas or infused into the pancreatic duct, with or without near-total pancreatectomy (as a means of inducing pancreatic ductal cell proliferation).. Groups of male Sprague-Dawley rats were exposed to varying doses of dimethylbenzanthracine (DMBA), methynitronitrosoguanidine, or ethylnitronitrosoguanidine either through direct implantation into the pancreas or infusion into the pancreatic duct. Near-total pancreatectomy was added in all groups except two DMBA implantation groups. Surviving rats were killed at 3, 6, 9, or 12 months, and the pancreata were evaluated histologically.. All three carcinogens caused pancreatic inflammation, ductal hyperplasia, atypia, and dysplasia beginning by 3 months and becoming more prominent at later time points. Only DMBA caused frequent invasive pancreatic ductal adenocarcinoma, which was first evident by 6 months. The prevalence of pancreatic cancer among DMBA-treated rats evaluated after 10 months was 39% (19 of 49). The addition of pancreatic resection did not enhance pancreatic cancer development.. Of the strategies tested, only direct implantation of DMBA into the rat pancreas frequently produces pancreatic cancer histologically similar to human ductal adenocarcinoma. The development of hyperplastic, atypical, and dysplastic changes preceding and accompanying carcinomas suggests that these lesions are preneoplastic. This model recapitulates the progression from normal to neoplastic epithelium and is likely to be useful for the study of morphologic and molecular mechanisms underlying the early stages of pancreatic carcinogenesis and for the investigation of novel diagnostic and therapeutic techniques.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinogens; Carcinoma, Ductal, Breast; Disease Models, Animal; Fibrosarcoma; Hyperplasia; Male; Methylnitronitrosoguanidine; Pancreatectomy; Pancreatic Ducts; Pancreatic Neoplasms; Rats; Rats, Sprague-Dawley; Sarcoma, Experimental

We have already reported that a combination of TNF-alpha and immunogenic variant was effective for immunotherapy of nonimmunogenic tumor. Immunogenic variants (A2 and A7) were obtained from nonimmunogenic fibrosarcoma (parent cell: 1767-3) by mutagen treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). In this study, we studied about the possibility of TNF-alpha gene immunotherapy for nonimmunogenic tumor. We established two types of TNF-alpha producing cell lines. One is TNF-alpha producing nonimmunogenic variant (1767 TR 2), and another is TNF-alpha producing immunogenic variant (A7 TR3). We compared them about the ability of producing specific immunity against parent tumor. TNF-alpha producing immunogenic variant (A7TR3) could induce strong specific immunity to parental cell compared to TNF-alpha producing nonimmunogenic variant (1767TR2). Immunogenicity of tumor is very important when we want to perform TNF-alpha gene immunotherapy against cancer.

Topics: Animals; Antigenic Variation; Fibrosarcoma; Genetic Therapy; Immunotherapy; Methylnitronitrosoguanidine; Mice; Mice, Inbred C3H; Mutagens; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Immunogenic variant was induced from the methylcholanthrene induced fibrosarcoma (MCA-F) by in vitro treatment with the mutagen 1-methyl-3-nitro-1-nitrosoguanidine (MNNG). D-10 tumor cell which was cloned from MNNG treated MCA-F tumor cell was rejected by normal syngeneic C3H-HeJ mice but not by 650 rad irradiated immunosuppressed mice. Host which had rejected D-10 tumor growth, rejected parental MCA-F tumor cells. But active immunotherapy using D-10 tumor cell against MCA-F tumor cells. But active immunotherapy using D-10 tumor cell against MCA-F tumor bearing mice was not successful. Cytotoxic T cell (CTL) established from D-10 immunized spleen cells showed D-10 specific cytotoxic activity and not lysed parent MCA-F cells. CTL clone C-E-6 showed specific cytotoxic and proliferative activity against D-10 cell. In vivo tumor neutralizing assay also showed its specificity against D-10 tumor cell. Active immunotherapy and adoptive immunotherapy using immunogenic variant, D-10 was not successful. D-10 tumor cell possesses very strong neoantigen induced by MNNG treatment and parental MCA-F antigen.

Topics: Animals; Antigens, Neoplasm; Female; Fibrosarcoma; Immunity, Cellular; Immunotherapy, Active; Immunotherapy, Adoptive; Methylcholanthrene; Methylnitronitrosoguanidine; Mice; Mice, Inbred C3H; Neoplasm Transplantation

During the course of malignant transformation of mammalian cells in vitro, a regular variation in chromosome number and structure is usually found. In order to elucidate the relationship between chromosomal changes and malignant expression, we isolated five clones from a malignant transformed Syrian hamster fibroblast cell line and analysed their biological characteristics, as well as chromosomal changes. A positive correlation between chromosome No 15 monosomy and the transformed and malignant phenotype was observed. We suggest that a suppression gene may be located on chromosome No 15, and its deletion or the loss of the chromosome may result in expression of the malignant phenotype.

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

The purpose of this study was to compare the frequency of generation and in vivo cross-reactivity of highly immunogenic (Imm+) clones induced in a single parental murine fibrosarcoma cell line MCA-F by 4 weekly treatments with either UV-B radiation, 1-methyl-3-nitro-1-nitrosoguanidine, or 5-aza-2'-deoxycytidine. These agents are believed to induce Imm+ variants by different mechanisms. The frequency of Imm+ variant generation was similar for the three different protocols, suggesting that the frequency of Imm+ generation was related more closely to the cell line than the inducing agent used. The strength of the immunogenic phenotype, however, was better correlated to the agent used, since 1-methyl-3-nitro-1-nitrosoguanidine yielded clones with the strongest immunogenicities. Three of four UV-B-induced Imm+ clones grew preferentially in chronically UV-irradiated syngeneic mice, a phenotype associated with UV-induced skin tumors. Cross-reactivity was tested with two Imm+ clones from each treatment group in a modified immunoprotection assay that selectively engendered antivariant, but not antiparental, immunity. Under these conditions each clone, except one, protected against itself. The clones displayed a complex pattern of cross-protection. Intervariant cross-protection was sensitive to the challenge dose, suggesting possible differences in the strengths of the cross-reacting immunities. Conversely, parental cross-protection was observed only with high immunizing multiplicities of Imm+ cells. The clones expressed the Imm+ phenotype in both C3H/HeN and C3H/HeJ mice, suggesting that expression of mammary tumor virus antigens did not account for the strong antitumor immune response. We also investigated whether the level of major histocompatibility complex class 1 or class 2 expression and immunogenic phenotype were correlated. Flow cytofluorography using haplotype-specific anti-Kk and anti-Dk monoclonal antibodies did not reveal a consistent difference in the constitutive or gamma-interferon-induced class 1 expression by Imm+ clones. However, we did observe a significant increase in the constitutive expression of IAk by most of the Imm+ variant clones. Together, these data demonstrate that in this system Imm+ variants engendered by a variety of mechanisms can express a range of cross-reactive tumor rejection neoantigens, independent of parental tumor antigens or major histocompatibility complex antigen expression.

Topics: Animals; Azacitidine; Cross Reactions; Decitabine; Female; Fibrosarcoma; H-2 Antigens; Methylnitronitrosoguanidine; Mice; Mice, Inbred C3H; Phenotype; Tumor Cells, Cultured; Ultraviolet Rays

The influence of allogeneic blood transfusions (BT) on experimental tumor growth was investigated in three syngeneic, one allogeneic and one autochthonous tumor model in the rat. Con A induced T-cell response, relative distribution of lymphocyte subsets using flow cytometry and cytotoxic antibodies were determined. No differences in take rate, induction time, incidence and growth rate of tumors were observed in the different models. A significant decrease of cell-mediated immunity and a significant increase of peripheral Ia-positive cells could be observed. The relative distribution of T-cell subsets showed no differences between BT-groups and controls.

Topics: Adenocarcinoma; Adjuvants, Immunologic; Animals; Benzo(a)pyrene; Blood Transfusion; Ethylnitrosourea; Fibrosarcoma; Methylnitronitrosoguanidine; Neoplasm Transplantation; Neoplasms, Experimental; Neuroma; Rats; Rats, Inbred Strains; Stomach Neoplasms; Transplantation Immunology

The purpose of the study was to investigate the immunological and biological consequences of neoantigen expression by immunogenic tumor variants (Imm+) following in vitro treatment with the mutagen 1-methyl-3-nitro-1-nitrosoguanidine. The weakly immunogenic murine fibrosarcoma MCA-F was used because we have previously characterized the tumor-specific transplantation antigen expressed by this tumor. Immunogenic variant clones were obtained at high frequency following four treatments with 1-methyl-3-nitro-1-nitrosoguanidine. The immunogenicity of the Imm+ clones was confirmed by their progressive growth in immunosuppressed C3H/HeN mice and their lack of growth in normal syngeneic (C3H/HeN mice. The immune response engendered in immunocompetent mice after a single immunization with viable Imm+ cells was tumor specific, completely protecting hosts against challenge with 10,000-fold the minimum tumorigenic dose of parental MCA-F cells, but not against 10 minimum tumorigenic doses of the non-cross-reactive tumor MCA-D. The strong cross-protection elicited by Imm+ neoantigens against the parental tumor-specific transplantation antigen was not observed when soluble extracts or isolated plasma membranes of Imm+ cells were used for immunization. Immunogenic variant cells inactivated using either mitomycin C or gamma-irradiation also demonstrated a significantly diminished immunoprotective activity against challenge with the parent tumor. However, inactivated Imm+ cells and their isolated plasma membranes still expressed sufficient neoantigen to completely protect mice against homotypic Imm+, but not parental challenge. These results suggest that (a) the MCA-F Imm+ variants express neoantigens capable of engendering a strong specific as well as cross-protective immunity against challenge with either the parent or the variant and (b) the associative recognition of neoantigen and TSTA that results in strong cross-protection against challenge with the parent tumor requires immunization with viable Imm+ cells for full expression of the immunogenic phenotype.

Topics: Animals; Antigens, Neoplasm; Cell Survival; Female; Fibrosarcoma; Histocompatibility Antigens; Immunization; Methylnitronitrosoguanidine; Mice; Mice, Inbred C3H; Mitomycins; Mutation; Vaccines

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

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

Antigenic variants were derived from a mutagen-treated, apparently nonimmunogenic fibrosarcoma of strain 2 guinea pigs. Fibrosarcoma line 107C3, originally induced by exposure of fetal cells to a chemical mutagen, was treated in vitro with the same mutagen. Cells that survived mutagen treatment were cloned, and the clones were tested for growth in soft agar, in conventional and immunosuppressed syngeneic guinea pigs, and in nude mice. At early passages after treatment, all clones tested in conventional syngeneic guinea pigs either failed to grow intradermally or grew temporarily and then regressed. At later passages after treatment, five of eight evaluable clones grew progressively; the characteristic of intradermal tumor growth followed by tumor regression (tum- or regressor) was a stable property of three of eight evaluable clones. The number of tumor cells required to produce progressively growing intradermal tumors in 50% of the animals (TD50) of the three tum- clones was at least 4 orders of magnitude greater than the TD50 of the parent fibrosarcoma. Tum- clones were not detected among 10 clones derived from the untreated parent tumor. Regressor clones formed colonies in soft agar and grew progressively in immunosuppressed syngeneic guinea pigs and nude mice. Regressor clones contained tumor transplantation antigens. Guinea pigs immunized with clones that grew and regressed rejected a challenge with the parent tumor when the dose of parent tumor cells was 1 to 3 times the TD50. Guinea pigs immunized by temporary growth of the parent tumor followed by excision of the local tumor and the regional lymph node did not reject a challenge with the parent tumor. These results confirm the results of experiments with murine tumors and extend the observations on tum- clones to the guinea pig. The results indicate that in guinea pigs it is possible by immunization with tumor cell variants derived from the mutagen-treated parent tumor to produce transplantation immunity to an apparently nonimmunogenic tumor.

Topics: Animals; Antigens, Neoplasm; Cell Division; Cell Line; Clone Cells; Fibrosarcoma; Genetic Variation; Guinea Pigs; Male; Methylnitronitrosoguanidine; Mice; Mice, Nude; Neoplasm Transplantation; Transplantation, Heterologous

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

Proliferation of granulation tissue on the inside of a subcutaneous (s.c.) air pocket was induced in rats by administration of 0.5 ml of 0.25% croton oil (granuloma pouch). Two days after induction of the granuloma, i.e., during the period of maximal cell growth, a single dose of 0.6 mg or 0.1 mg N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was administered into the pouch. Fibrosarcomas of various histopathological types developed in 87% of the rats receiving the high dose and in 64% of the low dose animals. The mean latency period was 47.5 and 51.7 weeks respectively. Only one local sarcoma developed in rats treated with 0.6 mg MNNG by the s.c. route, and no tumors were observed in the groups treated with 0.1 mg s.c. The appearance of local sarcomas in the granuloma pouch tissue is correlated with previously reported frequency of point mutations (OUAR and HGPRT-) induced in granuloma fibroblasts with the same doses of MNNG. Possible mechanisms explaining the marked enhancement of the carcinogenic effect of MNNG in the granuloma pouch assay are discussed.

Topics: Animals; Croton Oil; DNA; Dose-Response Relationship, Drug; Fibrosarcoma; Granuloma; Male; Methylnitronitrosoguanidine; Mutation; Rats; Sarcoma, Experimental

Carcinogens injected into the excretory canal of submandibular gland of Donryu rats revealed the following histologic changes in salivary glands. 20-Methylcholanthrene induced squamous cell metaplasia, fibrosis in the early stages, and "benign lymphoepithelial lesion"-like pattern after 3 months. Dense hyalinization occurred after 4-5 months with so-called "mixed tumor"-like pattern. In the later stages epidermoid carcinoma and fibrosarcoma were observed. 9, 10-Dimethylbenzanthracene caused degenerative change, metaplasia, fibrosis and cell infiltration, and later carcinoma and sarcoma appeared at a high rate. 4-Nitroquinoline-N-oxide led to dense hyalinization and so-called "mixed tumor"-like pattern was observed in many specimens. N-nitroso-N-methyl urethane and N-methyl-N-nitroso-N'-nitroguanidine revealed metaplastic changes, fibrosis and lymphoid infiltration. Scarlet red induced remarkable infiltration and aggregation of lymphoid cells, showing benign "lymphoepithelial lesion"-like pattern.

Topics: 4-Nitroquinoline-1-oxide; 9,10-Dimethyl-1,2-benzanthracene; Adenoma, Pleomorphic; Animals; Carcinogens; Carcinoma, Squamous Cell; Coloring Agents; Fibrosarcoma; Hemangioma; Injections; Male; Methylcholanthrene; Methylnitronitrosoguanidine; Nitrosomethylurethane; Rats; Rhabdomyosarcoma; Salivary Gland Neoplasms; Salivary Glands; Submandibular Gland

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