methylnitronitrosoguanidine and Skin-Neoplasms

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

This paper describes specific genetic changes involving chromosome 7 in mouse skin tumors, the most important consequence of which appears to be an alteration in the allelic balance of normal and mutant H-ras genes. The use of restriction-fragment-length polymorphisms in F1 hybrid mice demonstrates that trisomy of chromosome 7 is an early event preceding papilloma formation, and further events, such as mitotic recombination, seem to occur during progression to malignant carcinomas. There is some evidence of a tumor-suppressor locus situated on chromosome 7.

Topics: Alleles; Animals; Carcinoma; Chromosomes; DNA Damage; Gene Expression Regulation, Neoplastic; Genes, ras; Genes, Tumor Suppressor; Hybridization, Genetic; Methylnitronitrosoguanidine; Mice; Papilloma; Polymorphism, Restriction Fragment Length; Proto-Oncogene Proteins p21(ras); Recombination, Genetic; Skin Neoplasms; Trisomy

Inbred mouse strains that differing widely in their susceptibility to multistage skin carcinogenesis provide useful models for studying the genetic factors involved and advancing our understanding of the biochemical and molecular events associated with this process. The process of skin tumor initiation appears to be somewhat similar in various strains of mice, and most data in the literature suggest that differences in response to skin tumor promoters are a major determinant in controlling susceptibility to multistage skin carcinogenesis. A model system has been developed for examining the genetics of susceptibility to skin tumor promotion. The susceptibility to phorbol ester skin tumor promotion in crosses between DBA/2 and C57BL/6 mice is inherited as an incomplete dominant trait, and neither X-chromosome nor cytoplasmic genetic determinants appear to play a major role in determining susceptibility in these two inbred strains. In addition, two or more genetic loci contribute to the higher sensitivity of DBA/2 mice than C57BL/6 mice to TPA-induced skin tumor promotion. Further studies to characterize these genes will contribute greatly to our understanding of the mechanisms of phorbol ester skin tumor promotion. In addition, much work should now be directed at understanding the cellular, biochemical, and molecular mechanisms for differential responsiveness not only to phorbol esters but also to other classes of tumor promoters.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Cricetinae; Disease Susceptibility; Guinea Pigs; Methylnitronitrosoguanidine; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred DBA; Phorbol Esters; Rats; Skin Neoplasms; Species Specificity; Tetradecanoylphorbol Acetate

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Gene Expression Regulation; Genes, ras; Methylnitronitrosoguanidine; Mice; Mutation; Oncogenes; Restriction Mapping; Skin Neoplasms; Tetradecanoylphorbol Acetate; 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

The SENCAR mouse stock was selectively bred for eight generations for sensitivity to skin tumor induction by the two-stage tumorigenesis protocol using 7,12-dimethylbenz(a)anthracene (DMBA) as the initiator and 12-O-tetradecanoylphorbol-13-acetate (TPA) as the promoter. The SENCAR mouse was derived by crossing Charles River CD-1 mice with skin-tumor-sensitive mice (STS). The SENCAR mice are much more sensitive to both DMBA tumor initiation and TPA tumor promotion than CD-1, BALB/c, and DBA/2 mice. An even greater difference in the sensitivity to two-stage skin tumorigenesis is apparent between SENCAR and C57BL/6 mice when using DMBA-TPA treatment. However, the SENCAR and C57BL/6 mice have a similar tumor response to DMBA-benzoyl peroxide treatment, suggesting that TPA is not an effective promoter in C57BL/6 mice. The DBA/2 mice respond in a similar manner to the SENCAR mice when using N-methyl-N-nitro-N-nitrosoguanidine (MNNG)-TPA treatment. The SENCAR mouse model provides a good dose-response relationship for many carcinogens used as tumor initiators and for many compounds used as tumor promoter. When compared to other stocks and strains of mice, the SENCAR mouse has one of the largest data bases for carcinogens and promoters.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinogens; Carcinoma; Disease Models, Animal; Drug Evaluation, Preclinical; Methylnitronitrosoguanidine; Mice; Mice, Inbred Strains; Papilloma; Skin Neoplasms; Species Specificity; Tetradecanoylphorbol Acetate

Skin tumors chemically induced in mice have provided an important experimental model for studying carcinogenesis and for bioassaying carcinogenic agents. The information obtained from this model suggests that the events leading to tumor formation can be divided into at least two stages, initiation and promotion. A single small dose of carinogen produces initiation which appears to be irreversible. These initiating agents may have to be metabolically activated and can interact with cellular macromolecules. The extent to which they bind to DNA correlates well with their carcinogenicity. Increased DNA replication at the time of or during the first day after these agents have been applied appears to enhance carcinogenesis. Unlike initiation, promotion appears to be reversible and the promoting agents must be applied repeatedly before tumors are formed. Promoters interact with membranes, stimulate and alter genetic expression, and increase the rate of cell proliferation. The knowledge gained from these studies in mouse skin has immeasurably helped the entire field of chemical carcinogenesis. But efforts to determine the cellular and molecular mechanisms involved in the carcinogenic process, particularly in the skin, have been hampered by the difficulties of working on whole animals and by the special problems associated with the biologic and biochemical methods required for this target organ. Such problems, however, can be solved by the use of cell cultures of mouse epidermis which can metabolize and bind carcinogens just as is done in vivo. The fact that epidermal cells in vitro proliferate synchronously should facilitate the study of the relation between the cell cycle and carcinogenesis. These cells repair chemically induced DNA damage by at least two mechanisms, excision repair and base-specific repair. When epidermal cells in vitro are exposed to promoting agents, a proliferative response analogous to that in vivo is elicited, apparently mediated through control of polyamine metabolism. Neoplastic transformation has been induced in these cultures by known skin carcinogens.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Aryl Hydrocarbon Hydroxylases; Benzopyrenes; Carcinogens; Cell Division; Cells, Cultured; Chemical Phenomena; Chemistry; Croton Oil; Culture Media; Disease Models, Animal; DNA, Neoplasm; Drug Synergism; Kinetics; Methylnitronitrosoguanidine; Mice; Ornithine Decarboxylase; RNA, Neoplasm; Skin Neoplasms

The present survey deals with requirements the experimental-oncological models used in morphogenetic investigations should meet. Data on ways of inducing tumours of various organs the most suited for such investigations are presented. The available at present literature comprises data on different variants of morphogenesis of tumours; in a number of cases malignant neoplasms can develop against the background of an unchanged structure without previous alterations. Because of a contradictory character of the literature reports on morphogenesis of tumours, further investigations into the morphodynamics of the process of cancerogenesis are needed; at present, this may be successfully implemented if adequate models of the majority of tumour diseases in man are available. These studies are of importance for better understanding of pathogenesis or tumour growth and for ascertaining the concept of precancer changes.

Topics: 2-Acetylaminofluorene; 9,10-Dimethyl-1,2-benzanthracene; Aflatoxins; Animals; Azoxymethane; Benzopyrenes; Brain Neoplasms; Carcinogens; Cricetinae; Dimethylhydrazines; Dogs; Esophageal Neoplasms; Ethionine; Ethylnitrosourea; Female; Hematopoietic System; Intestinal Neoplasms; Liver Neoplasms; Lung Neoplasms; Male; Mammary Neoplasms, Experimental; Methylnitronitrosoguanidine; Methylnitrosourea; Mice; Neoplasms, Experimental; Nitrosamines; Nitroso Compounds; Nitrosoguanidines; o-Aminoazotoluene; p-Dimethylaminoazobenzene; Precancerous Conditions; Rats; Skin Neoplasms; Stomach Neoplasms; Urinary Bladder Neoplasms

Benzo[a]pyrene (B[a]P) is a ligand for the aryl hydrocarbon receptor (Ahr). After binding ligand, Ahr dimerizes with the aryl hydrocarbon receptor nuclear translocator (Arnt) protein, and the dimer upregulates the transcription of Cyp1a1, Cyp1b1 and other enzymes involved in the metabolic activation of B[a]P. Arnt null mice die in utero. Mice in which Arnt deletion occurs constitutively in the epidermis die perinatally. In the current study, mice were developed in which the Arnt gene could be deleted specifically in adult skin epidermis. This deletion had no overt pathological effect. Homozygosity for a null reduced nicotinamide adenine dinucleotide (phosphate): quinone oxidoreductase allele was introduced into the above mouse strain to render it more susceptible to tumor initiation by B[a]P. Deletion of Arnt in the epidermis of this strain completely prevented the induction of skin tumors in a tumor initiation-promotion protocol in which a single topical application of B[a]P acted as the tumor-initiating event, and tumor promotion was provided by repeated topical applications of 12-O-tetradecanoyl phorbol-13-acetate (TPA). In contrast, deletion of Arnt did not prevent the induction of skin tumors in a protocol also using TPA as the promoter but using as the initiator N-methyl-N'-nitro-N-nitrosoguanidine, whose activity is unlikely to be affected by the activity of Ahr, Arnt or their target genes. These observations demonstrate that Arnt is required for tumor initiation by B[a]P in this system.

Topics: Animals; Aryl Hydrocarbon Hydroxylases; Aryl Hydrocarbon Receptor Nuclear Translocator; Benzo(a)pyrene; Carcinogens; Cytochrome P-450 CYP1A1; Cytochrome P-450 CYP1B1; Epidermis; Gene Deletion; Methylnitronitrosoguanidine; Mice; Receptors, Aryl Hydrocarbon; Skin Neoplasms; 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

Hypermethylation of CpG sites within the promoter region of the O6-methylguanine-DNA methyltransferase (MGMT) gene occurs frequently in human cancer, preventing both MGMT expression and repair of alkylation damage. To assess the role of MGMT in the development of mouse skin tumors induced by initiation-promotion protocols, methylation of the MGMT promoter was examined in tumor DNA using methylation-specific PCR. To determine whether MGMT promoter methylation was affected by the tumor induction protocol, tumors were initiated by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or 7,12-dimethylbenz[a]anthracene (DMBA) and promoted by 12-O-tetradecanoylphorbol-13-acetate (TPA) or mezerein. Although the MGMT promoter was not methylated in normal skin, promoter methylation was found in 56 of 136 papillomas (41.2%) and in 19 of 37 squamous cell carcinomas (51.4%). When methylation of the MGMT promoter was compared in the 4 treatment groups, hypermethylation was found more frequently in tumors initiated by DMBA and promoted by mezerein, a protocol associated with a high frequency of malignant conversion. Methylation was found in some tumors as early as 5 weeks after initiation, but the methylation frequency increased with time. MGMT promoter methylation reduced MGMT expression as determined by immunohistochemistry. Although MGMT promoter methylation was not generally correlated with ras mutations, the frequency of MGMT methylation was higher in MNNG-initiated, mezerein-promoted papillomas with mutations in Ha-ras compared to papillomas with Ki-ras. Methylation of the MGMT promoter, associated with reduced MGMT expression, is found in nearly half of mouse skin tumors, but varies with both the tumor initiator and tumor promoter, and may be a key step in the progression from papillomas to carcinomas.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinogens; Carcinoma, Squamous Cell; Diterpenes; DNA Methylation; Gene Expression Regulation, Neoplastic; Genes, ras; Humans; Methylnitronitrosoguanidine; Mice; Mutation; O(6)-Methylguanine-DNA Methyltransferase; Papilloma; Promoter Regions, Genetic; Skin; Skin Neoplasms; Tetradecanoylphorbol Acetate

To determine whether chronic exposure to DMBA or MNNG in combination with or without UVB exposure would induce skin carcinomas in swine.. Eight gilts were exposed to 100 mJ of UVB in their left side, allowed to recuperate, and divided into two groups. Each gilt received identical high doses (DMBA 50 microM; MNNG 250 mM), low doses (DMBA 500 nM; MNNG 2.5 mM), carrier (DMSO), or nothing added treatments in the UVB and non-UVB sides. Animals were exposed weekly for 30 weeks and skin samples collected at 10, 20, and 30 weeks from initiation of exposure. An additional sample was collected 16 weeks following cessation of exposure. All samples were scored for dermal morphology, including intracellular epidermal edema, intercellular epidermal edema, papillary dermal edema, perivascular infiltrates, pyknotic stratum basale cells, collagen necrosis, and epidermal-dermal separation, and the data were analyzed by ANOVA. MNNG and UVB light had a significant effect on epidermal thickness and the number of cell layers. The greatest increase in epidermal thickness occurred from 20 weeks to 30 weeks in the UVB plus MNNG treatment. Treatment with MNNG resulted in intracellular and intercellular epidermal edema, dermal edema, and dermal inflammation at both the low and high doses of MNNG. In contrast, all the morphological evaluations of the DMBA treatments were less severe than the MNNG.. Our findings show that although chronic exposure to MNNG and DMBA, with or without UVB exposure, caused severe to mild dermatopathological changes, neither resulted in the development of skin carcinomas. These results indicate that at least with respect to responses to DMBA and MNNG, the swine model mimics more closely the responses seen in human skin.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Analysis of Variance; Animals; Cocarcinogenesis; Female; Methylnitronitrosoguanidine; Neoplasms, Radiation-Induced; Skin; Skin Neoplasms; Swine; Ultraviolet Rays

Previous studies demonstrated that repetitive application of cigarette smoke condensate (CSC) to 7,12-dimethylbenz[a]anthracene (DMBA)-initiated SENCAR mouse skin for 29 weeks at doses of 10, 20 and 40 mg "tar"/application results in time- and dose-dependent dermal tumor formation. To evaluate CSC-induced tumor promotion in other mouse skin models, male DBA/2 mice were treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (300 microg) or DMBA (75 or 150 microg) followed by promotion with 1R4F CSC at concentrations ranging from 9 to 45 mg "tar"/application. Both MNNG and DMBA have previously been shown to adequately initiate tumor development. Study end-points included clinical signs, body weights, and mass tracking. Neither the DMBA-initiated/acetone-promoted control groups, nor DMBA-initiated/CSC-promoted groups produced grossly observable skin tumors. For MNNG-initiated groups, a total of four tumors were observed. Based on these findings, it would appear the DBA/2 mouse was unresponsive to CSC dermal tumor promotion. It is not possible, based on the study design employed, to determine the underlying basis for the apparent resistance exhibited by this mouse strain to CSC-induced tumor promotion.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Body Weight; Male; Methylnitronitrosoguanidine; Mice; Mice, Inbred DBA; Mice, Inbred SENCAR; Nicotiana; Polycyclic Aromatic Hydrocarbons; Skin Neoplasms; Smoke

Elevated polyamine levels as a consequence of targeted overexpression of ornithine decarboxylase (ODC) to murine skin enhance susceptibility to tumorigenesis in this tissue. A possible mechanism for the enhanced susceptibility phenotype is an increased sensitivity of tissues with elevated polyamine levels to the mutagenic action of carcinogens. To test this hypothesis, a transgenic mouse model containing the Big Blue transgene and also expressing a K6/ODC transgene was developed. Incorporation of the K6/ODC transgene into the Big Blue model did not affect the spontaneous lacI mutant frequency in either skin or epidermis of the double-transgenic mice. After skin treatment with single doses of either 7,12-dimethylbenz[a]anthracene or N-methyl-N'-nitro-N-nitrosoguanidine, however, the mutant frequency was significantly increased in the skin of double-transgenic Big Blue;K6/ODC mice compared to Big Blue controls. The increases in mutant frequency were clearly due to ODC transgene activity, since treatment of mice with the ODC inhibitor, alpha-difluoromethylornithine, completely abolished the difference in mutant frequencies between double-transgenic and Big Blue mice. These results demonstrate that intracellular polyamine levels modulate mutation induction following carcinogen exposure.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinogens; Cocarcinogenesis; Methylnitronitrosoguanidine; Mice; Mice, Transgenic; Mutagenesis; Mutation; Ornithine Decarboxylase; Ornithine Decarboxylase Inhibitors; Polyamines; Skin Neoplasms

The skin tumor initiators N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and 7,12-dimethylbenz[a]anthracene (DMBA) differ in effectiveness when tumor formation is promoted by 12-O-tetradecanoylphorbol-13-acetate (TPA). Even at high doses, MNNG is less effective, producing fewer benign and malignant tumors with a longer latent period. In DMBA-initiated skin, 10 wk of TPA promotion produced a maximal tumor response. With MNNG, 20 wk of TPA promotion was required, producing nearly four times as many papillomas as 10 wk of promotion. Promotion of MNNG-initiated skin with mezerein induced the appearance of very rapidly-growing papillomas within 5 wk, 3 wk earlier than the first TPA-promoted papillomas. Thus, MNNG may induce a novel mutation resulting in a population of initiated cells that respond especially well to mezerein. Since ras mutations are common in experimental tumors in many tissues, we determined the frequency of activating mutations in the Ha-ras, Ki-ras, and N-ras oncogenes. Activating Ha-ras mutations were present in essentially all DMBA-initiated tumors and about 70% of MNNG-initiated tumors. No N-ras mutations were found in tumors lacking other ras mutations. Surprisingly, 41% of the papillomas arising in the first 11 wk in MNNG-initiated, mezerein-promoted mice bore mutations in codon 12 of the Ki-ras oncogene. Activating Ki-ras mutations were also found in more than 60% of squamous cell carcinomas and 40% of keratoacanthomas. Although mutations in Ha-ras are frequently detected in mouse skin tumors, mutations in Ki-ras are rare. This is the first report of mutated Ki-ras in skin tumors from mice initiated by MNNG.

Topics: Animals; Base Sequence; Carcinogens; Codon; Diterpenes; DNA Primers; Female; Genes, ras; Methylnitronitrosoguanidine; Mice; Mutagens; Mutation; Papilloma; Skin Neoplasms; Terpenes

The mechanism of action of fotemustine, a relatively new chloroethylnitrosourea, was evaluated in human melanoma cells in order to assess its potential as an agent for hyperthermic limb perfusion. Fotemustine was more toxic to O6-alkylguanine methyl transferase (AGT) deficient (Mer-) cells than Mer+ cells, implicating AGT as a major determinant of resistance. Mer+ cells derived from Mer- cell lines following exposure to the monofunctional alkylating metabolite of dacarbazine (DTIC) were also resistant to fotemustine. Mer status did not influence the replication of fotemustine-damaged adenovirus 5, whereas virus treated with the monofunctional alkylating agent N-methyl-N1-nitro-N-nitrosoguanidine (MNNG) was replicated much more efficiently by Mer+ cells. This suggests that the initial O6-alkylated product, if not immediately repaired, rearranges to form DNA crosslinks which cannot be repaired by AGT. Replication of a control virus was not affected by treating the cells with fotemustine, indicating that the drug acted primarily on DNA rather than at epigenetic levels. Fotemustine generally produced a G2-M block in the cell cycle, most strikingly in Mer- cells at low, minimally toxic concentrations; MNNG and high doses of fotemustine induced S phase arrest. Concurrent hyperthermia (41.5 degrees C for 1 h) increased the toxicity of fotemustine in some cell lines. Fotemustine decomposed in culture medium in two phases; the first was complete within 5 min and was most marked in Mer+ cells. The results suggest that fotemustine may be suitable for isolated limb perfusion in melanoma, with the potential for overcoming resistance by including inhibitors of AGT.

Topics: Adenoviridae; Antineoplastic Agents; Carcinogens; Cell Cycle; Cell Survival; DNA, Neoplasm; Drug Evaluation; Humans; Hyperthermia, Induced; Melanoma; Methylnitronitrosoguanidine; Nitrosourea Compounds; O(6)-Methylguanine-DNA Methyltransferase; Organophosphorus Compounds; Perfusion; Skin Neoplasms; Tumor Cells, Cultured; Virus Replication

Secreted phosphoprotein 1 (spp1), the gene encoding osteopontin (OPN), is expressed in many human carcinomas, although its in vivo functions remain unclear. To delineate the role of OPN during tumor progression, we have subjected OPN null mutant mice to repeated applications of a mutagen/carcinogen to induce cutaneous squamous cell carcinoma. OPN null animals exhibited accelerated tumor growth and progression and had a greater number of metastases per animal compared with wild-type animals. However, metastases in the OPN null animals were significantly smaller than in controls. When injected into nude mice, the growth of OPN null tumor lines and the same lines engineered to reexpress spp1 recapitulated the growth differences observed in the progression study. These differences in tumor growth inversely correlated with the degree of macrophage infiltration. Slower-growing, OPN-producing tumors contained significantly more macrophages, although a higher proportion were mannose receptor positive, a characteristic of differentiated resting macrophages. In vitro, OPN null cell lines displayed decreased survival at clonal density compared with OPN-producing lines, an observation consistent with the smaller metastases of the OPN null mice. Overall, we provide evidence for a model where host-derived OPN acts as a macrophage chemoattractant, whereas tumor-derived OPN is able to inhibit macrophage function and enhances the growth or survival of metastases.

Topics: Animals; Carcinogens; Carcinoma; Carcinoma, Squamous Cell; Cell Count; Cell Division; Disease Progression; Female; Male; Methylnitronitrosoguanidine; Mice; Neoplasm Proteins; Osteopontin; Papilloma; Phenotype; Sialoglycoproteins; Skin Neoplasms

Analysis of the expression of a number of known genes in cultured human cells has revealed UVB-induced changes that may be specific for melanocytic cells. The response of c-fos, p53 and HIV-LTR reporter constructs to UVB and UVC was reduced in MM96L melanoma cells compared to HeLa. Cell cycle arrest produced by UVA, gamma radiation, cisplatin or the antimetabolite deoxyinosine differed from that of UVB. Cell cycle analysis after multiple doses of UVB raised the possibility that UVB-induced pRb depletion could result in increased mutation and thus enhanced tumourigenesis of irradiated melanocytes in skin subjected to a defined pattern of UVB exposure. To extend the analysis of gene expression in cultured melanocytic cells to uncharacterised genes, promoter trap cell clones containing unknown genes 'tagged' by a beta-galactosidase reporter construct were generated from MM96L cells. Altered gene expression in clones treated with a panel of DNA-damaging agents was quantitated by measurement of beta-galactosidase activity. Of the clones containing 'tagged' endogenous promoters induced by UVB, 52% were induced only by UVB and not by other DNA-damaging agents (cisplatin, N-methyl-N-nitro-nitrsoguanidine, fotemustine). One third of the clones were also activated by TPA suggesting that general DNA damage responses involving PKC are activated less frequently than unique pathways of gene activation. Overall, 60% of the 50 clones that responded to the panel of agents were induced by only one of the agents, indicating that a high proportion of genes are induced by agent-specific mechanisms. In the long term, promoter trapping may allow the full repertoire of UVB-inducible genes to be characterised.

Topics: Antineoplastic Agents; beta-Galactosidase; Cell Cycle; Cisplatin; Demecolcine; Genes, fos; Genes, p53; HeLa Cells; HIV Long Terminal Repeat; Humans; Hydroxyurea; Inosine; Melanocytes; Melanoma; Methylnitronitrosoguanidine; Neoplasms, Radiation-Induced; Nitrosourea Compounds; Organophosphorus Compounds; Proto-Oncogene Proteins c-fos; Recombinant Proteins; Skin Neoplasms; Tetradecanoylphorbol Acetate; Transfection; Tumor Cells, Cultured; Ultraviolet Rays

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

In multistage tumorigenesis models, ornithine decarboxylase (ODC) is usually dysregulated at some point during tumor promotion, an early stage of carcinogenesis. To address the question whether constitutive overexpression of ODC would be a sufficient condition for tumor promotion, mice with high levels of ODC expression targeted to epidermal keratinocytes were used in skin tumorigenesis experiments. Transgenic mice with ODC targeted to hair follicle keratinocytes were much more sensitive than littermate controls to initiation with a single low dose of carcinogen; in fact, such mice no longer required treatment with tumor promoters for tumors to develop. Targeting ODC overexpression to both interfollicular and follicular keratinocytes did not further enhance tumor yield. Our results suggest that most, if not all, target cells for chemical carcinogens in the skin reside in hair follicles, and ODC overexpression is sufficient to activate such cells to expand clonally to form epidermal tumors.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Age Factors; Animals; Animals, Newborn; Benzo(a)pyrene; Hair Follicle; Immunohistochemistry; Methylnitronitrosoguanidine; Mice; Mice, Inbred C57BL; Mice, Transgenic; Ornithine Decarboxylase; Putrescine; Skin; Skin Neoplasms; Spermidine; Tetradecanoylphorbol Acetate; Time Factors

To develop mouse strains useful for studies of susceptibility and resistance to the induction of skin tumors, three new inbred SENCAR strains were independently derived by random inbreeding of outbred SENCAR mice. Characterization of these mice for sensitivity to skin tumor development indicated that mice of all three strains displayed increased sensitivity to initiation by 7,12-dimethylbenz[a]anthracene (DMBA), urethane, or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA). Promotion by mezerein as well as carcinogenesis by repeated treatment with DMBA or MNNG produced papillomas with a high frequency of conversion to squamous cell carcinomas (SCCs). Compared with outbred SENCAR mice, development of both squamous papillomas and carcinomas was increased at least two-fold by all protocols tested. The F1 hybrid between SENCARA/Pt males and resistant BALB/cAnPt females was resistant to the induction of both papillomas and SCCs after initiation by 2 microg of DMBA and promotion by 20 weekly applications of 2 microg of TPA. Papillomas developed in all of the SENCARA/Pt mice, none of the BALB/cAnPt mice, and 12% of the F1 progeny. Thus, at these doses of initiator and promoter, resistance was incompletely dominant in the F1 hybrid. However, the responsiveness of the F1 mice could be increased substantially by increasing the dose of the promoter.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinogens; Carcinoma, Squamous Cell; Cocarcinogenesis; Disease Susceptibility; Diterpenes; Female; Male; Methylnitronitrosoguanidine; Mice; Mice, Inbred BALB C; Mice, Inbred SENCAR; Papilloma; Phenotype; Sensitivity and Specificity; Skin Neoplasms; Terpenes; Urethane

Skin papillomas and squamous cell carcinomas (SCCs) are induced in mice by tumor initiation with a carcinogen followed by tumor promotion with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). These usually arise from preneoplastic lesions characterized by epidermal proliferation and hyperplasia, dermal edema, and inflammation. To evaluate the role of polypeptide growth factors in chemically induced skin carcinogenesis, we used transgenic mice carrying the cDNA for a TGF-beta related molecule, bone morphogenetic protein-4 (BMP-4), under the control of the regulatory elements of the cytokeratin IV* gene in a skin carcinogenesis protocol. Control non-transgenic littermates and BMP-4 transgenic mice were treated with a single dose of a carcinogen, N-methyl-N'-nitrosoguanidine (MNNG), and biweekly with the tumor promoter TPA for 9 months. In control littermates TPA induced epidermal hyperproliferation, atypia with "dark" cells, and dermal inflammation, resulting in papillomas and SCCs in 13 of 26 animals tested. In BMP-4 transgenic mice, TPA treatment induced the expression of the BMP-4 transgene in interfollicular epidermis but only minimal epidermal thickening, hyperproliferation, and inflammation were noted after the initial dose of TPA. Furthermore, the mitotic indices in transgenic epidermis after 9 months of TPA treatment were significantly lower than the corresponding indices from untreated transgenic epidermis. Consequently, none of the 22 transgenic animals tested developed papillomas or SCCs. In conclusion, we have shown that the TPA induced expression of the BMP-4 transgene blocks proliferation and inflammation in skin, steps that are critical to the subsequent formation of papillomas and SCCs and we characterized an inducible promotersystem which expresses polypeptides in interfollicular epidermis after exogenous stimulation.

Topics: Animals; Bone Morphogenetic Proteins; Bromodeoxyuridine; Carcinoma, Squamous Cell; Cell Division; Epidermis; Methylnitronitrosoguanidine; Mice; Mice, Transgenic; Papilloma; Proteins; Skin; Skin Neoplasms; Tetradecanoylphorbol Acetate; Transforming Growth Factor beta

Cell line SCR722 was derived from adult SENCAR mouse epidermal cells initiated in culture by treatment with the carcinogen N-methyl-N'-nitro-N-nitrosoguanidine and selection for foci proliferating in medium with calcium levels that induce terminal differentiation in normal cells. Expansion of one of these foci and two additional cell clonings produced cell line SCR722, which was near-tetraploid and formed normal skin when grafted to athymic nude mouse hosts. However, unlike normal keratinocytes, SCR722 cells fail to suppress papilloma formation when grafted along with papilloma cell line SP-1. For optimum growth in culture, SCR722 cells required fibroblast-conditioned medium and 0.5 mM Ca2+. SCR722 cells had a wild-type c-Ha-ras gene but had lost their requirement for conditioned medium in culture and produced dysplastic papillomas in grafts when transduced with the v-Ha-ras gene. SCR722 cells stably expressing the v-fos gene produced normal epidermis in grafts, but when these cells were transduced with the v-Ha-ras gene, they produced carcinomas. Clones with greater expression of the transfected v-fos gene had a more invasive phenotype in vivo. These results indicate that carcinogen treatment of epithelial cells can result in an altered but nontumorigenic phenotype that may be at risk for becoming a more advanced neoplastic state with additional genetic alterations.

Topics: Animals; Base Sequence; Cell Line; Evaluation Studies as Topic; Genes, fos; Genes, ras; Methylnitronitrosoguanidine; Mice; Mice, Inbred SENCAR; Mice, Nude; Molecular Sequence Data; Phenotype; Ploidies; Skin; Skin Neoplasms; Skin Physiological Phenomena; Transduction, Genetic

By analysis of skin tumors from F1 hybrid mice we demonstrated that the genetic events that occur during tumor progression depend on the type of chemical carcinogenesis protocol used to induce tumor growth. More than 95% of tumors induced by initiation with 7,12-dimethylbenz[a]anthracene (DMBA) and promotion with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) exhibited mutations in Ha-ras and trisomy of chromosome 7. Carcinomas induced with multiple DMBA treatments had a lower frequency of alterations on chromosome 7 (50%), but only in tumors with Ha-ras mutations, and had a much wider spectrum of alterations, including trisomy, mitotic recombination, deletion, and gene duplication. Carcinomas induced with multiple N-methyl-N'-nitro-N-nitrosoguanidine treatments only rarely exhibited alterations on chromosome 7 (8%), even if they contained mutant Ha-ras. More frequent numerical alterations of chromosome 11 were also seen in TPA-promoted tumors (23%) than in tumors induced by multiple carcinogen treatments (8%). These results show that postinitiation events are nonrandom and fit a model in which promoting agents induce numerical chromosomal alterations but in which mutagens cause more directed mutational events.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Chromosome Aberrations; Chromosome Deletion; Genes, ras; Methylnitronitrosoguanidine; Mice; Mutation; Polymorphism, Restriction Fragment Length; Skin Neoplasms; Tetradecanoylphorbol Acetate; Trisomy

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

The sensitivity of outbred SENCAR mice and inbred SENCAR (SSIN) mice to multistage carcinogenesis was studied. Tumors were induced using either 7,12-dimethylbenz[a]anthracene or N-methyl-N'-nitro-N-nitrosoguanidine as initiators and 12-O-tetradecanoylphorbol-13-acetate or benzoyl peroxide as promoting agents. Although the number of papillomas per mouse was higher in SSIN than in outbred SENCAR mice, the number of carcinomas observed in the SSIN strain was significantly lower regardless of the initiator or promoter used. It was also observed that the expression of markers of premalignant progression (i.e., dysplasia, expression of keratin K13, and loss of keratin K1 expression) was markedly suppressed in SSIN papillomas. After 50 wk of promotion with 12-O-tetradecanoylphorbol-13-acetate, the pattern of expression of K13 and K1 in SSIN mice was comparable to the pattern observed in outbred SENCAR mice after 10 to 20 wk of promotion with 12-O-tetradecanoylphorbol-13-acetate. It was also observed that 67% of the tumors induced in SSIN mice by initiation with 7,12-dimethylbenz[a]anthracene exhibited a mutation in codon 61 of the Ha-ras-1 gene. This latter finding suggests that the differences observed in tumor progression between the inbred strain and the outbred stock are not related to a genetic alteration in the Ha-ras-1 gene but rather to an independent event that we have postulated to involve a putative suppressor gene. The data reported here suggest that the putative gene(s) that confers susceptibility to tumor promotion was segregated from the gene(s) involved in tumor progression during selection and inbreeding of the SENCAR mouse stock.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Benzoyl Peroxide; Carcinoma, Squamous Cell; Female; Genes, ras; Keratins; Methylnitronitrosoguanidine; Mice; Mutation; Papilloma; Skin Neoplasms; Species Specificity; Tetradecanoylphorbol Acetate

The effects of palm carotene on chemical carcinogenesis was studied. Palm carotene suppressed mouse epidermal ornithine decarboxylase activity induced by glycocholic acid. In a two-stage mouse epidermal carcinogenesis experiment using 7,12-dimethylbenz(a)anthracene as the initiator, glycocholic acid as the 1st stage promoter, and mezerein as the 2nd stage promoter, palm carotene inhibited the promoting activity of glycocholic acid. Furthermore, in N-ethyl-N'-nitro-N-nitrosoguanidine-induced mouse duodenal carcinogenesis, 0.05% of palm carotene given in drinking water decreased the percentage of tumor-bearing mice significantly.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinogens; Carotenoids; Diterpenes; Duodenal Neoplasms; Female; Glycocholic Acid; Male; Methylnitronitrosoguanidine; Mice; Mice, Inbred C57BL; Mice, Inbred ICR; Ornithine Decarboxylase; Skin Neoplasms; Terpenes

The induction of micronuclei (MN) by incubation with different mutagenic agents was tested in diploid fibroblast cultures obtained from 15 probands with constitutively high MN rates (15.75-77.25 MN/500 cells; average 36.27 +/- 17.60 MN) and 15 probands (controls) with low MN rates (4-13.75 MN/500 cells; average 8.97 +/- 2.73 MN). In order to find out whether fibroblast cultures of individuals with increased spontaneous MN levels exhibit an increased sensitivity to various agents with different genotoxic mechanisms, we studied the induction of MN in these cell cultures by ultraviolet (UV) irradiation, mitomycin C (MMC), N-methyl-N-nitro-N-nitrosoguanidine (MNNG) and benzo-(a)pyrene-diol-expoxid (BPDE). In addition, we tested aphidicolin (APC), a polymerase alpha inhibitor, which is a potent inducer of common fragile sites. Probands with spontaneously high MN showed an significantly increased sensitivity to UV (p less than or equal to 0.005), MMC (p less than or equal to 0.005), and BPDE (p less than or equal to 0.005). No significant differences were found for MNNG and APC as compared to controls.

Topics: 7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; Aphidicolin; Cells, Cultured; Fibroblasts; Humans; Melanoma; Methylnitronitrosoguanidine; Mitomycin; Mutagens; Reference Values; Skin; Skin Neoplasms; Ultraviolet Rays

Ultraviolet irradiation can systemically enhance subsequent skin cancer induction by benzo[a]pyrene, methylcholanthrene, or UV radiation. The present study was designed to determine whether UVB irradiation influences host susceptibility to the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Female C3H/HeJ mice were exposed dorsally to UVB radiation from banks of 6 Westinghouse FS40 sun lamps. The mice received a total UV dose of approximately 8.1 x 10(5) J m-2 over a 15-week period. After termination of UVB treatments, ventral tumors were induced by 4 applications of 30 mumol of MNNG at 8-day intervals. At 20 weeks after the first MNNG treatment, UVB-irradiated mice had 7-fold more MNNG-induced, ventral tumors than did the unirradiated control mice (P = 0.026, Wilcoxon rank sum test). Ventral application of MNNG after cessation of dorsal UVB exposure, but before UV tumor appearance, did not influence photocarcinogenesis. These results demonstrate that UV irradiation can systemically decrease host resistance to tumor induction by the methylating agent, MNNG.

Topics: Animals; Female; Methylnitronitrosoguanidine; Mice; Mice, Inbred C3H; Neoplasms, Experimental; Neoplasms, Radiation-Induced; Skin Neoplasms; Time Factors; Ultraviolet Rays

Squamous cell carcinomas (SCC) of the mouse skin were produced by three different protocols of chemical carcinogenesis, i.e., complete carcinogenesis with 7,12-dimethylbenz(a)anthracene (DMBA) two-stage carcinogenesis with DMBA as initiator, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) as promoter and three stage carcinogenesis with DMBA, TPA and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) as third-stage agent or progressor. Tumors were sequentially studied at weeks 38-52 of treatment. Although no significant differences in the rate of appearance of gamma-glutamyl transpeptidase (GGT) could be seen, a larger number of SCC produced by complete carcinogenesis protocols were GGT-negative. This coincided with the higher grade of malignancy of these tumors as evaluated by histopathology. In general terms high-grade tumors were seen more frequently in the complete carcinogenesis experiment than in the other two protocols. SCC produced by complete carcinogenesis also exhibited a markedly higher DNA index than the SCC from the other experimental groups. All three protocols were very effective in producing late metastasizing tumors, and no significant differences could be established in the incidence of spontaneous lung metastasis. This shows that, contrary to general knowledge, if adequately observed for more than 40 weeks, SCC of the murine skin is able to metastasize in the lung in approximately 30% of cases. Nevertheless, complete carcinogenesis-induced SCC were usually of higher histological grade, a proportion of these were GGT-negative and produced more multiple or diffuse metastases than the tumors induced by the multistage protocols.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinoma, Squamous Cell; DNA, Neoplasm; Female; gamma-Glutamyltransferase; Lung Neoplasms; Methylnitronitrosoguanidine; Mice; Neoplasm Metastasis; Skin Neoplasms; Tetradecanoylphorbol Acetate; Time Factors

Mammary and skin tumors induced in rodents by N-methyl-N-nitrosourea treatment have a G:C to A:T transition mutation in codon 12 of H-ras, probably resulting from alkylation of O6 of guanine by the carcinogen. This codon contains two guanines (5'-GGA-3'), but mutations are observed only in the central base pair of this codon. The same selectivity for mutations of -GGA-sequences has also been observed in Escherichia coli. It is known that the central G in the sequence GGA is a preferred site for alkylation, but the magnitude of chemical selectivity is insufficient to provide a complete explanation for the biological observation which is still unexplained. We have measured accurate rates of repair by the E. coli and gene O6-alkylguanine-DNA-alkyltransferase of an O6-methylguanine in various positions in chemically synthesized 15-base pair DNA duplexes having the H-ras sequence. The rate of repair varied 25-fold, depending on the sequence flanking the methylguanine. An O6-methylguanine in position 2 of codon 12 was the least well repaired. The combination of this slow repair and sequence selectivity in alkylation appears to be the explanation for the selective mutation of this position. Using an antibody to probe the accessibility of the O6-methyldeoxyguanosine, it was shown that the rate of repair is a reflection of the conformation of the sequence containing the alkylated base, because the avidity constants between antibody and O6-methylguanine were also dependent on the sequence flanking the methylguanine, with the most rapidly repaired O6-methylguanines being those most easily bound by the antibody.

Topics: Animals; Bacterial Proteins; Base Composition; Base Sequence; Codon; DNA Repair; Escherichia coli; Escherichia coli Proteins; Genes, ras; Guanine; Kinetics; Mammary Neoplasms, Experimental; Methylnitronitrosoguanidine; Molecular Sequence Data; Nucleic Acid Conformation; Nucleic Acid Denaturation; O(6)-Methylguanine-DNA Methyltransferase; Oligodeoxyribonucleotides; Rats; Skin Neoplasms; Thermodynamics; Transcription Factors

A number of mouse skin tumors initiated by the carcinogens N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methylnitrosourea (MNU), 3-methylcholanthrene (MCA), and 7,12-dimethylbenz[a]anthracene (DMBA) have been shown to contain activated Ha-ras genes. In each case, the point mutations responsible for activation have been characterized. Results presented demonstrate the carcinogen-specific nature of these ras mutations. For each initiating agent, a distinct spectrum of mutations is observed. Most importantly, the distribution of ras gene mutations is found to differ between benign papillomas and carcinomas, suggesting that molecular events occurring at the time of initiation influence the probability with which papillomas progress to malignancy. This study provides molecular evidence in support of the existence of subsets of papillomas with differing progression frequencies. Thus, the alkylating agents MNNG and MNU induced exclusively G ---- A transitions at codon 12, with this mutation being found predominantly in papillomas. MCA initiation produced both codon 13 G ---- T and codon 61 A ---- T transversions in papillomas; only the G ---- T mutation, however, was found in carcinomas. These findings provide strong evidence that the mutational activation of Ha-ras occurs as a result of the initiation process and that the nature of the initiating event can affect the probability of progression to malignancy.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Base Sequence; Carcinogens; Codon; DNA, Neoplasm; Female; Genes, ras; Immunoblotting; Methylcholanthrene; Methylnitronitrosoguanidine; Methylnitrosourea; Mice; Mice, Inbred Strains; Molecular Sequence Data; Mutation; Nucleic Acid Hybridization; Oligonucleotide Probes; Polymerase Chain Reaction; Skin Neoplasms

Appreciable yields of cutaneous mast cell tumors were induced in a two-stage skin carcinogenesis protocol comprising N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) initiation followed by 12-O-tetradecanoylphorbol-13-acetate (TPA) promotion in 4 of 5 strains of mice. Only female mice of each of the 5 strains were studied. The incidences of benign and/or malignant lesions differed considerably between strains; 27% in DBA/2, 22% in BDF1, 11% in BALB/c, 10% in CDF1 and 0% in C57BL/6 mice and no mast cell tumors were detected in any of the strains when treated with the initiator alone. First found in a DBA/2 mouse at week 50, most tumors were observed after 100 weeks of promotion, and were usually small in size (less than 2 mm in diameter) and predominantly located within the corium, although they occasionally extended into the subcutaneous tissue. Histologically, the benign mast cell tumors were composed of non-encapsulated, well circumscribed densely packed sheets of discrete cuboidal or rhomboid cells. Metachromatic granules were clearly visible in the cytoplasm by Toluidine Blue staining. Two of the tumors induced in DBA/2 mice were diagnosed as malignant mast cell tumors on the twin bases of cellular atypia and deep infiltration into the muscular layer. The cutaneous mast cell tumors were constantly accompanied by subepidermal mast cell aggregations which were also commonly observed in tumor-free skin of mice receiving the initiation-promotion procedure.

Topics: Animals; Cell Aggregation; Cocarcinogenesis; Female; Mast Cells; Mast-Cell Sarcoma; Methylnitronitrosoguanidine; Mice; Mice, Inbred Strains; Skin Neoplasms; Species Specificity; Tetradecanoylphorbol Acetate

A single topical treatment of mouse skin with the potent tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) results in transient inductions of a variety of genes. Based on the time courses of their inductions, these genes can be classified into two main groups: "early" response genes whose mRNA expression reaches a maximum 0.5-2 h after TPA treatment and "secondary" response genes whose mRNA expression is maximal 4 h or more after treatment. The nuclear oncogenes c-fos, c-myc, and c-jun belong to the early response group, whereas the metallothionein, osteopontin, and urokinase genes belong to the secondary response group. The steady-state expressions of these early and secondary response genes are all very low in normal skin, except that of c-jun, which is relatively high. Steady-state levels of expression and inducibility of these genes by TPA were not altered in initiated skin or in apparently normal skin during tumor promotion. We examined the expressions of these genes in papillomas and carcinomas produced by two-stage (initiator-promoter) and three-stage (initiator-promoter-initiator) protocols in mouse skin. Steady-state expression of the early responding nuclear oncogenes in papillomas and carcinomas was found to remain at the same low level as in normal skin. However, all the secondary responding genes were found to be expressed constitutively at high levels in these tumors. Elevated expressions of the genes for transforming growth factor alpha and beta were also observed in papillomas and to varying extents in carcinomas. These observations suggest that the regulatory machinery for transcription by the protein kinase C-mediated pathway through nuclear oncogenes is altered during the processes of tumor promotion and progression. The genes whose expression is elevated may be associated directly or indirectly with tumor promotion and progression.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Blotting, Northern; Carcinogens; Carcinoma; Female; Gene Expression; Metallothionein; Methylnitronitrosoguanidine; Mice; Mice, Inbred Strains; Osteopontin; Papilloma; Proto-Oncogene Proteins; RNA, Messenger; Sialoglycoproteins; Skin; Skin Neoplasms; Skin Physiological Phenomena; Tetradecanoylphorbol Acetate; Transforming Growth Factors; Tubulin; Urokinase-Type Plasminogen Activator

We have shown previously that the risk of tumor initiation, promotion, and progression in animals initiated with alkylating agents can be drastically altered by hyperthermia treatments. We show here that ionizing radiation can also alter the risk of tumor initiation by alkylating agents. Using a two-step skin tumorigenesis protocol in female SENCAR mice (initiation by MNNG, promotion with TPA), we exposed the dorsal skin of the mice to various doses of 90Sr/90Y beta radiation near the time of initiation. The radiation produced a dose-dependent reduction in the number of papillomas which appeared after TPA promotion, with about a 20% reduction in animals receiving 0.5 Gy surface dose just before initiation, about 50% reduction after 2.5 Gy, and greater than 80% at doses above 5 Gy. A dose of 2.5 Gy in animals initiated with DMBA produced no significant reduction. One skin hyperthermia treatment (44 degrees C, 30 min) along with radiation in MNNG-initiated animals partially blocked the protective effect of radiation and increased the papilloma frequency. Radiation (2.5 Gy) given either 6 days before or after MNNG initiation was less effective but still reduced papilloma frequency about 20%. In sharp contrast to the marked reduction in papilloma formation, these same animals showed no change in carcinoma frequency with any of the doses or schedules of beta radiation. MNNG initiation alone produced three types of initiated cells. One type, produced in low yield, was promotion-independent with a high probability of progression to a carcinoma and appeared unaffected by the radiation. A second type, produced in intermediate yield, was promotion-dependent and also had a high progression probability, but was likewise unaffected by the radiation. The third and most abundant type was promotion-dependent with a very low progression probability. Radiation exposure resulted in a decrease in the risk of an MNNG initiation event which led only to the third type of cell. The data therefore indicate that the risk of some, but not all, tumor-initiating events caused by alkylating agents can be reduced by an exposure to ionizing radiation.

Topics: Animals; Beta Particles; Carcinoma; Female; Methylnitronitrosoguanidine; Mice; Papilloma; Skin Neoplasms; Strontium Radioisotopes

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

Topics: Amino Acid Sequence; Base Sequence; Biomarkers, Tumor; Blotting, Northern; Blotting, Southern; Carcinogens; Carcinoma, Squamous Cell; DNA; Fibroblasts; Gene Expression Regulation; Genes, Dominant; Genes, ras; Methylnitronitrosoguanidine; Molecular Sequence Data; Neoplasms, Radiation-Induced; Papilloma; Plasmids; Proto-Oncogenes; RNA; Skin Neoplasms; Suppression, Genetic; Transfection

Carcinogen treatment of normal mouse epidermal cells causes some cells, if cultured under the appropriate conditions, to continue to proliferate instead of terminally differentiate, forming foci at 37 degrees C in medium with a calcium level above 0.1 mM. We have examined these Calcium (Ca)-resistant cells formed in the skin of SENCAR mice after treatment with the carcinogen initiator 7,12-dimethylbenz[a]anthracene (DMBA) followed by tumor promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA). Although in our previous studies TPA promotion initially increased the size but reduced the number of foci caused by the carcinogen initiator N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), TPA promotion of DMBA-treated mice increased the size but had no effect on the number of foci. Papillomas resulting from DMBA plus TPA treatment contained many rapidly growing Ca-resistant cells, corroborating our earlier results with MNNG. Permanent cell lines prepared from papilloma-derived foci formed squamous cell carcinomas in nude mice after relatively short periods in culture. These data provide further evidence that Ca-resistant cells may be papilloma (and perhaps carcinoma) precursors in vivo. In addition, since TPA tends to reduce the number of early Ca-resistant cells caused by MNNG but not by DMBA, this may at least partially explain why treatment with DMBA plus TPA is much more effective in producing papillomas in SENCAR mice than is treatment with MNNG plus TPA.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Cell Differentiation; Epidermal Cells; Female; Methylnitronitrosoguanidine; Mice; Papilloma; Skin Neoplasms; Tetradecanoylphorbol Acetate

The in vivo behavior of cell cultures derived from normal and carcinogen-treated mouse epidermis was studied by implanting the cultures in a s.c. vascularized bed protected by a silicone chamber. Cells derived from normal adult mouse epidermis as well as cells derived from tumor-promoter-treated skin were unable to grow in these systems. Conversely, cell lines derived from skin initiated with single doses of N-methyl-N'-nitro-N-nitrosoguanidine or 9,10-dimethyl-1,2-benzanthracene proliferated in these chambers, reforming an epithelial structure. The type of structure in the chambers varied, ranging from formation of almost normal epithelia to atypical invasive behavior. The variable in vivo behavior among the different cell lines may be attributed to the initiation agent, the number of passages of the cultures, random genetic events, the strain of mouse, or a combination of these factors. Most of the cell types used in this study and all the cell lines that were able to grow in these chambers were selected for resistance to Ca-induced terminal differentiation. However, resistance to terminal differentiation according to the Ca2+ switch does not always correlate with the ability to grow in the chambers, since cell lines derived from spontaneous foci of resistance failed to grow in this system. These studies showed some of the possibilities of the SC silicone chambers to study the histogenic potential of cell lines derived from carcinogen-treated epidermis. This system also appears suitable to study the complex relationship between epidermal cells and specialized (dermal) stroma.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Calcium; Cell Line; Epithelium; Methylnitronitrosoguanidine; Mice; Mice, Inbred BALB C; Skin; Skin Neoplasms; Skin Transplantation

A single hyperthermia treatment given near the time of initiation with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or 7,12-dimethylbenz[a]anthracene (DMBA) increased the number of initiated cells in the skin of SENCAR mice subjected to a two-stage tumorigenesis protocol. In animals subsequently promoted with 12-O-tetradecanoylphorbol-13-acetate (TPA), a 44 degrees C, 30-min hyperthermia treatment given just before, just after or 24 h before MNNG initiation increased the average papilloma frequency by 40-50%. In the groups of animals that received a hyperthermia treatment just before MNNG initiation, tumor latency was reduced by 40-60%. Treatment with MNNG in the absence of hyperthermia produced two classes of initiated cells. One type, formed in low yield, was independent of TPA promotion and formed tumors with a high probability of progression to malignancy. The other type was promotion dependent, and formed in relatively high yield but produced tumors with a probability of progression to carcinomas approximately 10-fold less than promotion-independent initiated cells. A single hyperthermia treatment given just before or just after MNNG initiation increased the yield of both promotion-dependent and promotion-independent initiated cells, and consequently increased the yield of carcinomas. In animals given a single hyperthermia treatment 24 h prior to initiation (to induce thermotolerant skin cells), MNNG exposure resulted in an increased yield of promotion-dependent initiated cells but no change in the yield of promotion-independent initiated cells. Hyperthermia treatment of DMBA-initiated skin increased the yield of initiated cells (promotion-dependent) only when given just after exposure to the initiator. The extra initiated cells produced by hyperthermia treatment of MNNG or DMBA exposed skin had the same probability of progression to carcinomas as initiated cells produced by the same initiation in the absence of hyperthermia. As noted previously for DMBA-initiated mice, hyperthermia given at the time of each application of TPA promoter also suppressed the formation of papillomas initiated by MNNG. Only the promotion and progression of promotion-dependent initiated cells, and not of promotion-independent cells, was suppressed. The results show that a single hyperthermia treatment near the time of exposure to an alkylating agent increased the number of both promotion-dependent and promotion-independent initiated cells and, as a consequence, increased th

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinoma; DNA Repair; Female; Glutathione; Hyperthermia, Induced; Methylnitronitrosoguanidine; Mice; Papilloma; Skin Neoplasms; Tetradecanoylphorbol Acetate

Since current evidence suggests that the tumor promotion stage is a primary determinant in susceptibility to multistage carcinogenesis, we have characterized the genetics of susceptibility to phorbol ester skin tumor promotion in inbred mice. Susceptibility of hybrids (B6D2F1), between DBA/2 (sensitive) and C57BL/6 (resistant) parents, initiated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and promoted with 12-O-tetradecanoylphorbol-13-acetate (TPA) was similar to DBA/2 mice at doses of 13.6 nmol per mouse but clearly less when doses of 1.7-6.8 nmol per mouse were used. In addition, no significant differences were observed between male and female B6D2F1 mice in terms of tumor incidence although some differences were observed in tumor multiplicities between male and female F1 mice at the highest TPA dose. Reciprocal F1 mice initiated with DMBA (i.e. D2B6F1) were also responsive to TPA. Female D2B6F1 mice were of lower sensitivity at lower doses of TPA, compared to female DBA/2, a finding similar to that observed with B6D2F1 mice initiated with MNNG. Further analyses of the susceptibility of B6D2F2 and B6D2F1 X C57BL/6 backcross mice to TPA promotion indicated that more than one dominant genetic locus must account for the differences in promotion sensitivity between DBA/2 and C57BL/6 mice. To understand further the genes responsible for promotion sensitivity, histological evaluations were performed on DBA/2, C57BL/6 and B6D2F1 mice. Histological examination revealed that the epidermis of DBA/2 mice showed a marked hyperplasia and the presence of a much greater number of dark basal keratinocytes (DCs) compared with C57BL/6 mice 48 h after the last of four applications of TPA (doses greater than or equal to 3.4 nmol). A marked dermal infiltration of polymorphonuclear leukocytes (PMNs) was observed in DBA/2 mice, whereas little infiltration was observed in the skin of C57BL/6 mice. The hyperplasia in the skin of B6D2F1 mice was intermediate between DBA/2 and C57BL/6 mice at all TPA doses examined except the lowest dose (1.7 nmol), whereas the DC response, although significantly lower at doses of 6.8 nmol or below, was similar to DBA/2 mice at higher TPA doses (13.6 and 17.0 nmol). The infiltration of PMNs in the dermis of B6D2F1 mice was similar to or greater than DBA/2 mice at all doses of TPA tested.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Crosses, Genetic; Genes, Dominant; Male; Methylnitronitrosoguanidine; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Mice, Inbred Strains; Skin Neoplasms; Species Specificity; Tetradecanoylphorbol Acetate

Beta-carotene (BC) and canthaxanthine (CX), two carotenoids with and without pro-vitamin A activity, respectively, were found to help to prevent benzo[a]pyrene (BP)-induced skin carcinogenesis in the dark and BP photocarcinogenesis (UV 300-400 nm) when given as an oral supplement to female Swiss albino mice. The same experimental procedure was adapted to 8-methoxypsoralen (8-MOP) photoinduction of mammary carcinomas in mice. Here also, the two carotenoids were strongly antitumorigenic. Indeed, 8-MOP photomutagenesis, tested in S. typhimurium TA 102, appeared to depend on a two-step reaction, namely an oxygen-independent DNA-8-MOP photoadduct, followed by an oxygen-dependent second step, sensitive to carotenoids. This result suggests that dietary carotenoids (powerful antioxidant molecules) might prevent the carcinogenic risk caused by substances that are transformed into ultimate carcinogens by oxidative processes which are indirectly carcinogenic. Finally, to verify whether supplemental carotenoids can affect carcinogenesis where neither light excitation nor oxidative metabolic processes are involved, an experimental attempt was made on gastric carcinogenesis induced in rats by the direct carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The results demonstrate that supplemental carotenoids do not affect initiation and progression stages, but do prevent the progression stage of dysplasias to infiltrating gastric carcinomas. Thus, this provides strong presumptive evidence for oxygen radical involvement in the later stages of this neoplastic development, as recently reported in the literature. As far as mutagenicity in S. typhimurium is concerned, carotenoids do not exert, as expected, any protective effect on MNNG mutagenic activity. The above experimental data suggest that supplemental carotenoids, instead of sunscreen preparations, can be adopted by outdoor workers to prevent skin cancer. Accordingly, such natural antioxidants may be useful in human chemoprevention against neoplasias of the lung, breast, urinary bladder, and colon and rectum even after radical surgery.

Topics: Animals; Benzo(a)pyrene; beta Carotene; Canthaxanthin; Carcinogens; Carotenoids; Diet; Female; Free Radicals; Methoxsalen; Methylnitronitrosoguanidine; Mice; Mutagenicity Tests; Neoplasms, Experimental; PUVA Therapy; Skin Neoplasms

Alkaline elution was used to examine DNA single-strand breaks in cultured normal and carcinogen-altered mouse keratinocytes exposed to 12-O-tetradecanoyl phorbol-13-acetate and benzoyl peroxide. Seven cell lines derived from carcinogen-induced mouse skin papillomas and three cell lines derived from N-methyl-N'-nitro-N-nitrosoguanidine-treated non-tumor bearing mouse skin were resistant to phorbol ester-mediated DNA strand breaks after 6 or 24 h. Normal keratinocytes sustained strand breaks after 24 h but not after 6 h. Benzoyl peroxide induced extensive strand breaks in normal keratinocytes at both 6 and 24 h, and this was associated with marked cytotoxicity. In contrast, 9 of 10 cell lines showed complete or partial resistance to strand breaks following benzoyl peroxide exposure. It is proposed that differential resistance to DNA strand breaks and cytotoxicity among normal and carcinogen-altered keratinocytes provides the biological basis for the promoting action of benzoyl peroxide. Furthermore, sublethal DNA damage in preneoplastic or neoplastic keratinocytes may account for the potency of benzoyl peroxide in causing malignant conversion.

Topics: Animals; Benzoyl Peroxide; DNA Damage; Keratins; Methylnitronitrosoguanidine; Mice; Mice, Inbred BALB C; Papilloma; Peroxides; Skin; Skin Neoplasms

Chemical carcinogenesis in mouse skin has been divided into the process of initiation, promotion and progression. Recently we have shown that ionizing radiation acts as an initiator in this model system. In this paper we describe a three-stage experiment using ionizing radiation in the third stage of mouse skin carcinogenesis. CD-1 mice were initiated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) followed by biweekly promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA). After 20 weeks of promotion, the animals were treated with either acetone, TPA (twice a week for 2 weeks) or eight fractions of 1 MeV electrons (1 Gy/fraction over a period of 10 days). The conversion of papillomas to squamous cell carcinomas was 80% for animals treated with ionizing radiation compared with 25% for tumor-bearing animals treated with TPA. Ionizing radiation increased the number of cumulative carcinomas per group. The lack of an increase in the number of cumulative papillomas per group due to ionizing radiation suggests that the dose and fractionation protocol used in this study enhanced the progression of pre-existing papillomas.

Topics: Acetone; Animals; Carcinoma; Cocarcinogenesis; Female; Methylnitronitrosoguanidine; Mice; Neoplasms, Radiation-Induced; Papilloma; Skin Neoplasms; Tetradecanoylphorbol Acetate

Five doses of either N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or ethylnitrosourea (ENU) were tested as complete carcinogens, tumor initiators and tumor promoters in the SENCAR skin tumorigenesis model. As tumor initiators, MNNG-induced papillomas at all doses tested, while ENU was active from 10-40 mumol. As complete carcinogens, MNNG from doses of 0.5-5.0 mumol and ENU from doses of 10 mumol-40 mumol were potent inducers of both papillomas and carcinomas indicating that these agents are active as both tumor initiators and tumor promoters.

Topics: Animals; Carcinogens; Carcinoma; Cocarcinogenesis; Ethylnitrosourea; Female; Male; Methylnitronitrosoguanidine; Mice; Mice, Inbred Strains; Neoplasms, Experimental; Papilloma; Skin Neoplasms

The incidence of metastasis was evaluated in female SENCAR mice after induction of squamous cell carcinomas by repetitive applications of either benzo [a] pyrene (B [a] P) or N-methyl-N'-nitro-N-nitrosogaunidine (MNNG). Between 41 and 50 weeks 50% of the animals with carcinomas in the B [a] P group had metastases, whereas 20% had metastases in the MNNG group. Very few metastases were observed before 40 weeks of treatment. The major site of metastasis was the lungs; however, metastatic tumors were also found in lymph nodes, adrenal glands and kidneys.

Topics: Animals; Carcinoma, Squamous Cell; Female; Methylnitronitrosoguanidine; Mice; Neoplasm Metastasis; Skin Neoplasms; Time Factors

We have used an in vivo-in vitro approach to investigate the cellular aspects of two-stage skin carcinogenesis. Female SENCAR mice initiated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were promoted twice weekly with 12-O-tetradecanoylphorbol-13-acetate (TPA). Epidermal cultures from untreated or TPA-treated mice had few focus-forming cells resistant to calcium-induced terminal differentiation. Cultures from mice treated with MNNG alone formed numerous foci. Brief promotion (four TPA treatments) of MNNG-treated mice produced fewer but statistically larger foci, suggesting that TPA was selecting against more slowly growing cells. MNNG plus TPA-treated mice with very early papillomas produced more and larger foci than those due to MNNG treatment alone, suggesting that the papillomas may have comprised calcium-resistant cells. These cells may indeed be initiated cells since a permanent cell line arising after MNNG plus brief TPA treatment eventually formed histological papillomas in vivo. If calcium-resistant cells are initiated, then there were many more initiated cells in the skin (with or without TPA treatment) than papillomas expected, implying that either some initiated cells never formed papillomas, or that a significant accumulation of initiated cells had already occurred in the skin within 2 weeks of MNNG treatment. Subsequent TPA promotion of these cells apparently produced a toxic response that passively selected for more rapidly growing initiated cells, which eventually accumulated into papillomas.

Topics: Animals; Cell Differentiation; Cells, Cultured; Female; Methylnitronitrosoguanidine; Mice; Mice, Inbred Strains; Precancerous Conditions; Skin; Skin Neoplasms; Tetradecanoylphorbol Acetate

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

Mouse skin tumors were induced by a single topical application of 7,12-dimethylbenzanthracene (DMBA), followed by biweekly promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA). After 20 weeks of promotion, mice were treated twice weekly for 2 weeks with either ethylnitrosourea (ENU), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or TPA. Thereafter all groups were treated biweekly with TPA. The ENU-treated group had a higher percentage of animals with carcinomas and developed 217% more cumulative carcinomas per group than TPA-treated controls. The percentage of mice with carcinomas and the cumulative number of carcinomas per group in MNNG-treated mice was higher than TPA-treated controls but was less than ENU-treated mice. The ratio of cumulative carcinomas to cumulative papillomas in ENU treated, MNNG-treated and TPA-treated mice was 16%, 9% and 6%, respectively. Histological examination of tumors remaining at the termination of the experiment revealed the presence of keratoacanthomas, some of which stained positive for gamma-glutamyltransferase (GGT), in the ENU-treated and MNNG-treated, but not the TPA-treated groups. The fact that no new papillomas developed during the progression stage indicated that enhanced carcinogenesis resulted from the progression of pre-existing tumors. Enhanced progression of benign skin tumors in mice by only a few treatments of an agent may serve as a potential model for studies into the mechanisms and the inhibition of malignant progression. The model also allows for a comparison of the potency of agents in enhancing malignant progression.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Ethylnitrosourea; Female; gamma-Glutamyltransferase; Methylnitronitrosoguanidine; Mice; Papilloma; Skin Neoplasms; Tetradecanoylphorbol Acetate

The abilities of 4-O-methyl-12-O-tetradecanoylphorbol-13-acetate (4-O-methyl-TPA) and mezerein to promote the process of transformation were evaluated in cultures of C3H/10T1/2 mouse embryo fibroblasts treated with N-methyl-N'-nitro-N-nitrosoguanidine. Mezerein was found to be as potent as the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) for the promotion of focus formation, eliciting a promotion response at concentrations that ranged from 100 to 2500 ng/ml. 4-O-Methyl-TPA (25-2500 ng/ml) did not promote focus formation, but was mitogenic for confluent cultures. The effects of promoting and non-promoting compounds upon intercellular communication were then evaluated to determine if a rapid assay for the inhibition of communication might serve as a surrogate for the relatively long term, labor-intensive cell transformation assay. Inhibited intercellular communication, as measured by inhibition of [3H]uridine exchange between cells, appeared to correlate with the ability of phorbol related compounds to promote transformation. However, the potent promoter 2,3,7,8-tetrachlorodibenzo-p-dioxin did not inhibit [3H]uridine transfer. Inhibition of intercellular communication may thus be diagnostic of the promoting potential of phorbol-related compounds in C3H/10T1/2 cultures, but may not be an appropriate endpoint for the study of carcinogenic dioxins.

Topics: Animals; Carcinogens; Cells, Cultured; Dimethyl Sulfoxide; Diterpenes; Female; Fibroblasts; Methylnitronitrosoguanidine; Mice; Mice, Inbred C3H; Polychlorinated Dibenzodioxins; Pregnancy; Skin Neoplasms; Terpenes; Tetradecanoylphorbol Acetate; Time Factors; Uridine

The induction by chemical carcinogens of resistance to terminal differentiation in cultured mouse keratinocytes has been proposed to represent a cellular change associated with the initiation phase of skin carcinogenesis. Previous results with this culture model indicated that the number of differentiation-resistant foci was correlated with the dose and known potency for several chemical carcinogens. Assay conditions were optimized to provide quantitative results for screening a variety of carcinogens for their potency as inducers of foci resistant to terminal differentiation. Eight skin initiators of varying potency and from different chemical classes and ultraviolet light were studied for their activity to induce this alteration in cultured epidermal cells from newborn BALB/c mice. There was an excellent positive correlation for the potency of these agents as initiators in vivo and as inducers of altered differentiation in vitro. The induction of resistant foci was independent of the relative cytotoxic effects of each agent except where cytotoxicity was extensive and reduced the number of foci. The results support the hypothesis that initiation of carcinogenesis in skin results in an alteration in the program of epidermal cell differentiation. The results also suggest that the assay is useful for identifying relative potency classes (strong, moderate, weak) of initiating agents.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Calcium; Carcinogens; Cell Differentiation; Cells, Cultured; Methylnitronitrosoguanidine; Mice; Skin; Skin Neoplasms; Ultraviolet Rays

Primary adult mouse epidermal cells were maintained as a monolayer culture with a high proliferation rate in fibroblast-conditioned medium with low Ca2+ concentration (less than 0.1 mM). Terminal differentiation of the cultures was induced by raising the Ca2+ level in the medium above 0.1 mM. Treatment of adult mouse epidermal cells either in vivo or in vitro with 7,12-dimethylbenz(a)anthracene or N-methyl-N'-nitro-N-nitrosoguanidine yielded colonies which were resistant to terminal differentiation induced by Ca2+. The number of resistant colonies was dependent upon the dose of each carcinogen used whether exposure was in vivo or in vitro. Cultures derived from skin initiated in vivo with 7,12-dimethylbenz(a)anthracene, a strong initiator, resulted in more colonies than were derived from skin initiated with N-methyl-N'-nitro-N-nitrosoguanidine, a moderately potent initiator. Two mouse strains, BALB/c and SENCAR, which differ in sensitivity to skin carcinogenesis, yielded similar numbers of Ca2+-resistant colonies following carcinogen exposure. However, colonies developed spontaneously from untreated SENCAR cells (the sensitive strain), but not from BALB/c cells (the resistant strain). These results support the concept that cells resistant to terminal differentiation are initiated cells. The results also suggest that initiation may occur spontaneously in SENCAR skin, a finding consistent with the reported occurrence of tumors in mice of this strain receiving promoters without exogenous initiator.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Calcium; Cell Differentiation; Cell Division; Epidermal Cells; Male; Methylnitronitrosoguanidine; Mice; Mice, Inbred BALB C; Skin Neoplasms

The dynamic changes of chicken erythrocyte membranes induced by a series of N-methyl-N'-aryl-N-nitrosoureas (I X) were investigated in comparison with those of N-methyl-N-nitrosourea (MNU) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) by electron spin resonance spectroscopy. Both I X and MNNG increased the intensity of the less mobile lipid component (h1) during the incubation while MNU decreased it. The more mobile lipid component (h2), however, was not much influenced by these agents. A positive correlation was observed between the degree of the spectral alterations (h2/h1) caused by I-X compounds and their tumorigenic potency on mouse skin with the exception of the chloride derivative (I-C1). The present results suggest that the reactions of ultimate carcinogens with cell membranes may play an important role in the tumor promoting process.

Topics: Animals; Carcinogens; Chickens; Erythrocyte Membrane; In Vitro Techniques; Methylnitronitrosoguanidine; Methylnitrosourea; Mice; Nitrosourea Compounds; Skin Neoplasms; Solubility

The skin of white outbred rats was painted with solutions of N-methyl-N-nitrosourea (MNU), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), N-methyl-N,N'-dinitroguanidine and diazoacetic ester (DAAE). DAAE was administered to rats intravenously, intraperitoneally and subcutaneously as well. Skin tumours have appeared only in the experiments with MNU, MNNG and DAAE. The application of MNU and MNNG caused tumours of the skin in the site of application, and as for DAAE, it induced tumours in the remote places of the skin. Systemic methods of DAAE administration entailed mainly mammary tumours in female rats, but not those of the skin. Possible mechanisms of action of the compounds under study have been discussed.

Topics: Administration, Topical; Animals; Carcinoma, Basal Cell; Diazonium Compounds; Female; Guanidines; Injections, Intraperitoneal; Injections, Intravenous; Injections, Subcutaneous; Lung Neoplasms; Male; Methylnitronitrosoguanidine; Methylnitrosourea; Nitroso Compounds; Rats; Sarcoma, Experimental; Skin Neoplasms

Tumor initiation in CD-1 mice by benzo[a]pyrene (BaP) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was unaffected by topical pretreatment with 13-cis-retinoic acid (13-cis-RA). Likewise, anthralin-induced tumor promotion in SENCAR mice was unaffected by pretreatment with 13-cis-RA. These results suggest that the action of retinoids in preventing either tumor initiation or promotion is very carcinogen or cocarcinogen specific.

Topics: Animals; Anthralin; Benzo(a)pyrene; Benzopyrenes; Female; Isotretinoin; Methylnitronitrosoguanidine; Mice; Papilloma; Skin Neoplasms; Time Factors; Tretinoin

Mice of the inbred strain DBA/2 responded to a two-stage, initiation-promotion tumorigenesis protocol when high initiating doses (400 nmol/mouse) of 7,12-dimethylbenz[a]anthracene were utilized. They also responded when N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was used as the initiating agent. The tumor response in both cases was characterized by a rapid rate of tumor development with the maximal tumor responses reached on or before the 15th week of promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA). When DBA/2 mice were compared with SENCAR mice for promotion sensitivity following initiation with MNNG, the two mouse stocks responded with a nearly identical tumor response. C57BL/6 mice were essentially resistant to TPA promotion regardless of the initiator or the dose of initiator used. A preliminary study was conducted to determine how susceptibility to tumor promotion by TPA was inherited in F1 mice derived from DBA/2 (sensitive) and C57BL/6 (resistant) parents. The B6D2F1 mice were as sensitive as the DBA/2 parent, suggesting that susceptibility in these two inbred mouse strains is inherited as an autosomal dominant trait. The results show that these two inbred mouse strains may provide a model system for studying genetic factors controlling susceptibility to phorbol ester skin tumor promotion.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinogens; DNA; Female; Methylnitronitrosoguanidine; Mice; Mice, Inbred DBA; Mice, Inbred Strains; Phorbols; Skin Neoplasms; Species Specificity; Tetradecanoylphorbol Acetate

Solution of N-methyl-N-nitrosourea (MNU), diazoacetic ester (DAAE), N-methyl-N-nitro-N-nitrosoguanidine (MNNG), N-methyl-N,N'-dinitroguanidine, 4-dimethylaminoazobenzene (DAB), o-aminoazotoluene or 7,12-dimethylbenz(a)anthracene (DMBA) were applied to rat skin. Neoplasms (skin tumors) were found only in the experiments with MNU, DAAE, MNNG, DAB and DMBA, MNU, MNNG, DAB and DMBA induced tumours in the sites of skin painting. In experiments with DAAE distant skin neoplasms developed as well. Possible mechanisms of action of these compounds are discussed.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinoma, Squamous Cell; Diazonium Compounds; Female; Keratoacanthoma; Male; Methylnitronitrosoguanidine; Methylnitrosourea; o-Aminoazotoluene; p-Dimethylaminoazobenzene; Papilloma; Rats; Skin Neoplasms

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

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Cell Division; Dose-Response Relationship, Drug; Hyperplasia; Methylnitronitrosoguanidine; Mice; Mice, Inbred Strains; Phorbols; Rats; Rats, Inbred F344; Skin; Skin Neoplasms; Species Specificity; Tetradecanoylphorbol Acetate

SENCAR mice are markedly more susceptible to two-stage skin carcinogenesis than BALB/c mice. Studies were carried out to elucidate the basis for this sensitivity. It is not related to differences in the metabolism of polycyclic aromatic hydrocarbons (DiGiovanni et al., 1980) but appears to be determined by the target tissue, since when SENCAR skin was grafted onto nude mice they developed papillomas at a high frequency after initiation and promotion, whereas after grafting of BALB/c skin, no tumours developed. DNA repair capacity was studied in SENCAR and BALB/c epidermal cells in culture. Host cell reactivation, utilizing ultra-violet light-irradiated herpes simplex virus, was similar in cells of the two strains. SENCAR cells have a greater binding capacity for epidermal growth factor than BALB/c cells; however, the increased binding in response to retinoic acid and the rapid decrease after exposure to phorbol esters are similar in the two strains. Spontaneous expression of endogenous proviral DNA sequences for xenotropic-type C RNA viruses occurs more readily in BALB/c epidermal cells than in those of SENCAR. The frequency of spontaneous differentiation-resistant foci in vitro (Kulesz-Martin et al., 1980) is greater in SENCAR than in BALB/c epidermal cells. These results suggest that susceptibility for skin carcinogenesis in SENCAR mice is determined by the target tissue itself and has no clear relation to DNA excision repair, endogenous virus complement or epidermal growth factor receptors.

Topics: Animals; Calcium; Cell Differentiation; DNA Repair; Epidermal Growth Factor; Methylnitronitrosoguanidine; Mice; Mice, Mutant Strains; Neoplasms, Experimental; Skin Neoplasms; Virus Replication

The carcinogenic activities of a number of directly acting methylating and ethylating agents have been compared by mouse skin painting in acetone solution. Nitrosomethylurethane and nitrosoethylurethane failed to induce tumors after greater than 60 weeks treatment. Nitrosomethylurea was somewhat more effective than nitrosoethylurea, as measured by the longer latent period than nitrosoethylurea, as measured Nitrosomethylnitroguanidine, by the same measure, was a weaker carcinogen than nitrosoethylnitroguanidine at both dose levels used (0.02 M and 0.008 M); the latter compound was the most potent skin carcinogen of those examined. There was no significant difference in carcinogenic effectiveness when the alkyl group of the nitrosoureas or the nitronitrosoguanidines contained deuterium instead of hydrogen, which supports the concept that alkylation of cellular macromolecules by the intact alkyl group is responsible for carcinogenesis by these compounds.

Topics: Administration, Topical; Alkylating Agents; Animals; Deuterium; Ethylnitrosourea; Methylnitronitrosoguanidine; Methylnitrosourea; Mice; Neoplasms, Experimental; Nitroso Compounds; Nitrosoguanidines; Nitrosomethylurethane; Skin Neoplasms

In order to define factors which determine susceptibility to chemical carcinogenesis, mice sensitive (SENCAR) and resistant (BALB/c) to epidermal carcinogenesis were studied under several treatment conditions for sensitivity to initiation by 7,12-dimethylbenz(a)anthracene or N-methyl-N'-nitro-N-nitrosoguanidine and promotion by 12-O-tetradecanoylphorbol-13-acetate. In newborns of both strains, topical application of initiator was much less effective than in adults. However, initiation by i.p. injection of 7,12-dimethylbenz(a)anthracene is at least as effective in newborns as in adults, which may indicate that topically applied carcinogen is not delivered effectively to target cells in newborns. Thus, newborn epidermis can respond to 7,12-dimethylbenz(a)anthracene as well as adult epidermis when the initiator is appropriately administered. SENCAR mice are much more sensitive than are BALB/c mice to both initiators, which suggests that enhanced metabolic activation of hydrocarbon carcinogens by SENCAR mice is unlikely to account for their sensitivity. Newborn male SENCAR's developed approximately 50% more papillomas than did females in all groups. BALB/c newborn mice developed so few tumors that a meaningful comparison of sensitivity of males and females could not be made. Thus, the increased sensitivity of SENCAR's was apparent regardless of route of administration of initiator or the age or sex of the mice. SENCAR mice also developed a significant number of papillomas and squamous cell carcinomas with 12-O-tetradecanoylphorbol-13-acetate promotion in the absence of an exogenous initiator. Therefore, the skin of SENCAR mice may contain an initiated population of cells capable of responding to tumor promoters.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Age Factors; Animals; Benz(a)Anthracenes; Carcinoma, Squamous Cell; Cocarcinogenesis; Dose-Response Relationship, Drug; Methylnitronitrosoguanidine; Mice; Mice, Inbred Strains; Papilloma; Skin Neoplasms

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

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; Croton Oil; DNA; DNA Repair; DNA Replication; Drug Synergism; Female; Methylnitronitrosoguanidine; Mice; Neoplasms, Experimental; Papilloma; Skin; Skin Neoplasms; Time Factors