Showing papers on "Carcinogenesis published in 1980"

A survey of transformation markers in differentiating epidermal cell lines in culture.

Abstract: Primary mouse epidermal cells underwent spontaneous malignant transformation in culture. Twelve malignant epidermal cell lines were established which produced squamous cell carcinomas in syngeneic hosts. These lines were used to define criteria for recognizing transformed epidermal cells in vitro . Growth in suspension in agar, agarose, or Methocel was minimal for 11 of the lines. All lines tested retained specific epidermal antigens (pemphigus, pemphigoid, keratin) by indirect immunofluorescence, but keratin content was reduced when quantified by radioimmunoassay. Basal activity of ornithine decarboxylase and activity induced by the tumor promoter 12- O -tetradecanoylphorbol-13-acetate were variable among lines. All malignant lines as well as normal epidermal cells grew well at reduced extracellular calcium concentrations. When the extracellular calcium was elevated, normal cells ceased proliferation, terminally differentiated, and sloughed from the culture dish, while malignant cells continued to proliferate although they expressed differentiative functions. These results indicate that malignant transformation in epidermis is associated with a fundamental alteration in the program of terminal differentiation which allows some cells to escape the proliferative block and cell death which accompanies differentiation in normal keratinocytes. This alteration should be useful to select for transformants during the process of carcinogenesis in vitro .

Covalent binding of polycyclic aromatic compounds to mitochondrial and nuclear DNA.

Abstract: Since the pioneering work of the Millers it has become clear that most chemical carcinogens require metabolism to reactive electrophiles and then exhibit their carcinogenic potential by reacting chemically with, and modifying, cellular macromolecules. At first modification of proteins was considered most likely to be of importance in carcinogenesis. Later, Brookes and Lawley demonstrated that the extent of binding of several polycyclic hydrocarbons to DNA, but not to RNA or protein isolated from the skin of mice treated topically with these compounds, correlated with their known carcinogenic potency to this tissue. Mammalian cells, particularly mouse embryo cells, treated with chemical carcinogens have often been used, and DNA has been involved almost exclusively from whole cells. However, mitochondria possess unique DNA which accounts for 0.1-1% of the total DNA present in mammalian cells, and three studies have shown that carcinogenic alkylating agents modify the michondrial DNA by a factor about five times greater than the nuclear DNA from the same cells. We demonstrate here that with six polycyclic aromatic compounds, all of which require metabolic activation and bind to DNA to a much smaller extent than direct than direct-acting alkylating agents, the binding to mitochondrial relative to DNA is dramatically increased by a factor of nearly 50 to over 500.

Carcinogenesis : fundamental mechanisms and environmental effects

Abstract: Carcinogenicity of Polycyclic Aromatic Hydrocarbons: The Bay-Region Theory.- Inactivation of OX174 and SV 40 Viral DNA Replication by Diol Epoxide Derivatives of Carcinogenic Polycyclic Hydro-carbons.- Dihydrodiols and Diol-Epoxides in the Activation and Detoxification of Polycyclic Hydrocarbons.- Metabolically Generated Free Radicals from Many Types of Chemical Carcinogens and Binding of the Radicals with Nucleic Acid Bases.- Nucleophilicity of DNA. Relation to Chemical Carcinogenesis.- An Analysis of the Reactivities of Epoxide Rings in Some Cyclic Hydrocarbons.- Biological Effects of Specific Hydrocarbon-DNA Reactions.- The Effect of Base Modification on Fidelity in Transcription.- Modification of DNA by Carcinogenic Aromatic Amines In Vivo and In Vitro with Possible Promutagenic Consequences.- Bio-chemical Considerations of the Enzymology Associated with Quinone and Tetrol Formation During Benzo (a) pyrene Metabolism.- Stereochemical Aspects of Benzo (a) pyrene Metabolism and Biochemical Individuality in Human Cells.- Metabolic and Structural Requirements for the Carcinogenic Potencies of Unsubstituted and Methyl-Substituted Polycyclic Aromatic Hydrocarbons.- Formation and Inactivation of DNA-Binding Metabolites of Benzo (a) pyrene Studied with Isolated Cells and Subcellular Fractions.- Enzymic Control of Reactive Metabolites from Aromatic Carcinogens.- Aflatoxin-DNA Interactions: Qualitative, Quantitative and Kinetic Features in Relation to Carcinogenesis.- Structural Modifications and Specific Recognition by Antibodies of Carcinogenic Aromatic Amines Bound to DNA.- Immunological Detection and Quantification of DNA Components Structurally Modified by Alkylating Carcinogens (Ethylnitrosourea)..- Characteristics of Stages of Hepatocarcinogenesis.- Mechanisms of Genetic Susceptibility to Cancer.- Constitutive Uncoupling of Pathways of Gene Expression that Control Growth and Differentiation and the Molecular Mechanism of Carcinogenesis.- Amplification of Carcinogenesis by Non-Carcinogens - Diterpene Type Promoters and Models of Environmental Exposure.- Reduction of Human Exposure to Environmental N-Nitrosocarcinogens. Examples of Possibilities for Cancer Prevention.- Endogenous Carcinogenesis: Nitrate, Nitrite and N-Nitroso Compounds.- Neoplastic Transformation, Somatic Mutation, and Differentiation.- Oncogenic Transformation, Initiation, Promotion and Mutagenesis in C3H/10T1/2 Cells.- Dissection of the Early Molecular Events in the Activation of Lymphocytes by 12-0- Tetradecanoylphorbol-13-acetate.- Modulation of Adipose Conversion of BALB/c 3T3 Cells by Tumor Promotors.- Established Cell Cultures as Model Systems for Carcinogen Metabolism.- Regulation of the Expression of SV40 T Antigen in Stem Versus Differentiated Cells.- The Effect of 12-0- Tetradecanoyl-phorbol-13-acetate (TPA) on Cell Transformation by Simian Virus 40 Mutants.- The Use of Cell Cultures to Assay the Effects of Chemicals on Bone Marrow.- Structural Modifications and Their Effects on the Genetic Functions of DNA Generated by Interactions with Benro (a) pyrene Metabolites.- Neoplastic Transformation of Human Mutant Cells by a Tumor Promoter.- Use of Human Epidermal Keratinocytes in Studies on Chemical Carcinogenesis.- Studies on Why 12-0-Tetra- decanoyl-phorbol-13-acetate (TPA) Does Not Promote Epidermal Carcinogenesis of Hamsters.- Modulation of Repair of DNA Damages Induced by Nitrosamines.- DNA Repair in Human Cells Exposed to Combinations of Carcinogenic Agents.- Reactions of Aflatoxin B1, Damaged DNA In Vitro and In Situ in Mammalian cells.- The Role of DNA Repair in Preventing the Cytotoxic and Mutagenic Effects of Carcinogens in Human Cells.- Transformation of Diploid Human Fibroblasts by Chemical Carcinogens.- Monoclonal Antibodies in the Study of Human Cancer.- Oculo-Cutaneous and Internal Neoplasmas in Xeroderma Pigmentosum: Implications for Theories of Carcinogenesis.- The Induction, Expression and Modulation of Radiation Induced Oncogenesis In Vitro in Diploid Humand and Rodent Cells.- Progress in Cloning the Transforming Gene from Chemically- Transformed Mouse Cells.- Relationship Between Transformation and Mutation in Mammalian Cells.- Membrane and Other Biochemical Effects of the Phorbol Esters and Their Relevance to Tumor Promotion.- The Chemistry of Poly- cyclic Aromatic Hydrocarbon-DNA Adducts.- Index of Subjects.

Transforming genes of neoplasms induced by avian lymphoid leukosis viruses.

Abstract: Oncogenic avian retroviruses can be classified into three groups: sarcoma viruses, acute leukaemia viruses and lymphoid leukosis viruses (LLVs)1. Sarcoma and acute leukaemia viruses transform fibroblasts and/or haematopoietic cells in culture and induce tumours with short latent periods in infected birds. In contrast, LLVs do not transform cells in vitro and require long latent periods before formation of neoplasms in vivo. The most frequent neoplasm induced by LLVs is malignant lymphoma of the bursa of Fabricius, but LLVs also induce other neoplasms, including sarcomas, nephroblastomas and erythroblastosis2. The genomes of both sarcoma and acute leukaemia viruses contain specific genes responsible for viral oncogenicity1,3–5, whereas the genome of LLVs apparently includes only genes required for virus replication. The genetic basis for the low oncogenic potential of LLVs is therefore obscure. The present experiments indicate that LLV-induced tumours contain transforming genes that can be detected by transfection of NIH 3T3 mouse cells. These transforming genes are not linked to LLV DNA sequences, suggesting that oncogenesis by LLVs may result from indirect activation of cellular transforming genes.

Modification of Mutagenic Activity

Abstract: The detection of environmental mutagens is extremely important in monitoring the conditions under which humans are living. Mutagens in the environment will affect human germ cell DNA, resulting in the accumulation of mutated genes in populations, and will also affect the DNA of somatic cells, resulting in initiation of carcinogenesis and possibly also aging of somatic cells.

Differential repair of O(6)-methylguanine in DNA of rat hepatocytes and nonparenchymal cells.

Abstract: Chronic administration of several chemical carcinogens to laboratory animals induces a variety of tumours which arise from specific cell populations within the liver. In the rat, diethylnitrosamine induces hepatocellular carcinomas1, dimethylnitrosamine induces both angiosarcomas2 and hepatocellular carcinomas3, vinyl chloride primarily induces angiosarcomas4, and 1,2-dimethylhydrazine induces malignant haemangioendotheliomas5,6. One of the principal mechanisms thought to be involved in initiating carcinogenesis is the alkylation of specific sites on DNA, such as the O6 position of guanine7. Previous investigations of alkylation and repair have, however, analysed DNA prepared from whole liver. This approach does not localize alkylation or repair capacity in the different cell types which give rise to neoplasia. Furthermore, although hepatocytes account for more than 90% of the liver's mass, they only comprise 60-70% of its cells8. The nonparenchymal cell (NPC) population, which consists almost entirely of endothelial and Kupffer cells, accounts for the remaining 30–40% and contains 10–20% of the DNA. Therefore, we decided to investigate the alkylation and repair of O6-and 7-methylguanine in the target and non-target cells following oral administration of 1,2-dimethylhydrazine. We report here that although initial alkylation was slightly less in NPCs, removal of O6-methylguanine was significantly slower. This led to a preferential accumulation of O6-methylguanine in NPC 24 h after administering a second daily dose. In contrast, 7-methylguanine decreased at similar rates, resulting in a 28-fold greater O6-methylguanine/7-methylguanine ratio in the target cell population.

Lipids and carcinogenesis.

Abstract: Experiments with animals and epidemiological data on human populations have provided evidence that high fat diets increase the incidence of certain types of cancer, such as breast cancer and colon cancer. High fat diets enhance mammary tumorigenesis in rats only when the fat contains a certain minimal level of essential fatty acids. Dietary fat appears to act as a promoter rather than affecting initiation of mammary tumors. It may do this by producing a more favorable environment for development and growth of tumor cells, either by changing the hormonal environment, by altering the properties of cell membranes thorugh changes in their lipid composition, or by other mechanisms, such as alterations in immune responses to tumor cells. The effect of dietary fat on colon cancer may be related to increased production and excretion of bile acids, some of which have been shown to be promoters of intestinal cancer in animals. It may be possible to utilize this knowledge of the effects of dietary fat on carcionogenesis to develop new methods for prevention and treatment of breast and colon cancer.

Efficiency of the adaptive response of Escherichia coli to alkylating agents

Abstract: When cultures of Escherichia coli are exposed to a low level of the alkylating agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) they accumulate mutations for about 20 min and then become resistant to further mutagenesis by that level of MNNG1. This ‘adaptive response,2 has been shown to be due, at least in part, to induction of the rapid repair of O6-alkyl-guanine3–5 which appears to be the main mutagenic and carcinogenic lesion produced by simple alkylating agents6,7. A similar kind of repair has been demonstrated in the livers of rats exposed to nitrosamines8, and this presumably helps to protect animals against carcinogenesis by the various alkylating agents that are widespread in our environment. It seemed important, therefore, to find out just how effectively such adaptive responses can control mutation rates.

Membrane and other Biochemical Effects of the Phorbol Esters and their Relevance to Tumor Promotion

Abstract: The pleiotropic effects of TPA and related phorbol esters on a variety of cell cultures provide important clues to the process of tumor promotion and the multistep nature of carcinogenesis. These effects can be divided into three categories: 1) mimicry of transformation in normal cells, and enhancement of transformation by chemical carcinogens or oncogenic viruses; 2) modulation (inhibition or induction) of differentiation; and 3) membrane and receptor effects. Recent evidence suggests that TPA acts by binding to specific high affinity cell surface membrane receptors and that this then leads to rapid alterations in the composition of membrane phospholipids. Presumably, these changes in the lipid matrix of cell membranes produce signals or mediators which lead to the subsequent cytoplasmic and nuclear effects of TPA. Thus, whereas the critical target in the action of initiating carcinogens appears to be cellular DNA, the critical target of the phorbol ester tumor promoters appears to be cell membranes. As a unifying concept of two-stage carcinogenesis, we postulate that during the initiation phase in carcinogenesis the covalent binding of carcinogens to DNA induces a host response somewhat analogous to that of the SOS response in bacteria. However, in mammalian cells this response results in abberations in the commitment of the target cells. This may involve ordered events, for example, gene transpositions, rather than random point mutations. Tumor promoters, via their effects on growth, gene expression and differentiation, enhance the selective outgrowth of Initiated cells and induce them to express their newly acquired but previously dormant committed state. Thus, initiation and promotion parallel events during normal development and differentiation, but during carcinogenesis the new cell populations are aberrant in terms of specialized functions and growth control.

Inhibition of carcinogen-induced chromosomal aberrations by an anticarcinogenic protease inhibitor

Abstract: It was hypothesized that chemicals- and radiation-induced carcinogenesis might require at least two specific chromosomal events that must coincide within a single target cell: (i) induction of chromosomal changes, possibly mutations, that are recessive and therefore latent in diploid somatic cells and (ii) aberrant mitotic segregation events that will convert the heterozygous cell, created by the first process, into a homozygous or hemizygous cell through chromosomal rearrangements. Hence, we tested the prediction that an inhibitor of induced carcinogenesis may inhibit one or both of these chromosomal events by studying the effects of antipain, a protease inhibitor and known inhibitor of carcinogenesis, on N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced mutagenesis, chromosomal aberrations, sister chromatid exchanges, and cell killing in V79 Chinese hamster cells. We show that antipain inhibited MNNG-induced chromosomal exchanges and all other chromosomal aberrations exclusively. This results leads us to postulate that MNNG-induced DNA lesions cause chromosomal aberrations which arise through an antipain-sensitive cellular process, that some chromosomal rearrangement is a rate-limiting step in carcinogenesis, and that mutagenesis alone, if required, is not sufficient to accomplish carcinogenesis.

Mammary gland cell-mediated mutagenesis of mammalian cells by organ-specific carcinogens.

Abstract: Rat mammary gland cells have been used to activate chemical procarcinogens to mutagenic compounds in a culture system. Mutagenesis was tested in Chinese hamster V-79 cells that were cocultured with the mammary cells. The locus mutations tested for were resistance to ouabain and resistance to 6-thioguanine. Mammary cells were separated into several fractions. Fractions enriched in either epithelial or stromal cells could both mediate mutagenesis. The mutation frequency related to the density of the mammary cells. The mammary carcinogen 7, 12-dimethylbenz( a )anthracene exhibited a dose-dependent enhancement of mutation frequency and cytotoxicity when added to the cocultures, whereas the hepatocarcinogen aflatoxin B 1 did not. This system may be useful in examining some of the mechanisms of organ-specific carcinogenesis and also may act as a screening system for carcinogenic environmental contaminants.

Microsome-mediated Covalent Binding of 1,2-Dichloroethane to Lung Microsomal Protein and Salmon Sperm DNA

Abstract: In order to determine whether the covalent binding of the carcinogen 1,2-dichloroethane to macromolecules is dependent on microsomes or cytosol, microsomes and cytosol from lungs of C57BL/6 × C3H/He F1 (hereafter called B6C3F1) mice and Osborne-Mendel rats were incubated with [1,2-14C]dichloroethane and salmon sperm DNA. 1,2-Dichlorothane binds covalently to microsomal protein and DNA only in the presence of microsomes, whereas cytosol has insignificant metabolic activation. The binding to macromolecules was significantly higher in the presence of native microsomes than denatured microsomes. The interaction of 1,2-dichloroethane with DNA was enhanced following pretreatment of the animals with phenobarbital and 3-methylcholanthrene. On the other hand, glutathione reduced the binding. The binding of 1,2-dichloroethane to lung microsomal protein of B6C3F1 mice and to DNA was three and five times higher, respectively, than that of Osborne-Mendel rat lung microsomal proteins. 1,2-Dichloroethane interacts 85% and 100% more with protein and DNA, respectively, in the presence of microsomes obtained from lung than from liver of B6C3F1 mice. These results suggest a correlation between the microsomally mediated binding and species and organ susceptibility to 1,2-dichloroethane-induced tumorigenesis.

Multistage chemical carcinogenesis in mouse skin.

Abstract: Skin tumors in mice can be induced by the sequential application of a subthreshold dose of a carcinogen (initiation phase) followed by repetitive treatment with a noncarcinogenic tumor promoter. The initiation phase requires only a single application of either a direct-acting carcinogen or a procarcinogen which has to be metabolized before being active; it is essentially an irreversible step which probably involves a somatic cell mutation as evidenced by a good correlation between the carcinogenicity of many chemical carcinogens and their mutagenic activities. There is a good correlation between the skin-tumor-initiating activities of several polycyclic aromatic hydrocarbons (PAH) and their ability to bind covalently to epidermal DNA. Results from our laboratory as well as others suggest that “bay region” diol-epoxides are the ultimate carcinogenic form of PAH carcinogens. Potent inhibitors and stimulators of PAH tumor initiation appear to affect the level of the PAH diol-epoxide reacting with specific DNA bases. Recent data suggest that the tumor-promotion stage involves at least 3 important steps: (l) the induction of embryonic-looking cells (dark cells) in adult epidermis; (2) an increased production of epidermal prostaglandins and polyamines; (3) sustained proliferation of dark cells. Retinoic acid specifically inhibits step 2, whereas the anti-inflammatory steroid fluocinolone acetonide is a potent inhibitor of steps 1 and 3. The mechanism and the importance of a specific sequence for each step in chemical carcinogenesis in mouse skin will be discussed in detail.

Polyoma large tumor antigen is not required for tumorigenesis mediated by viral DNA

Abstract: The arrangement of viral DNA sequences in a hamster cell line derived from a tumor induced by a recombinant plasmid DNA preparation containing the entire polyoma virus genome was examined. In the recombinant plasmid employed, viral DNA sequences specifying the large species of polyoma tumor antigen but not the small and middle tumor antigens were interrupted by the insertion of plasmid DNA at the EcoRI restriction endonuclease site. Blot-hybridization analyses of tumor cell DNA indicated that the "joints" linking viral and plasmid DNAs in the original recombinant plasmid used in animal inoculation had been preserved. Integration into the hamster cell genome had apparently occurred within plasmid DNA sequences. These results indicate that polyoma large tumor antigen is not required for tumorigenesis mediated by viral DNA.

In vitro generation of cytotoxic lymphocytes against radiation- and radiation leukemia virus-induced tumors. III. Suppression of anti-tumor immunity in vitro by lymphocytes of mice undergoing radiation leukemia virus-induced leukemogenesis.

Abstract: Adult C57BL/6 mice exposed to fractionated irradiation or inoculated with the radiation leukemia virus (RadLV), develop high incidence (80-100%) of lymphatic leukemias within 3-6 mo. RadLV-induced lymphomas can elicit cytotoxic responses in vitro in lymphocytes of preimmunized syngeneic mice, a reaction that is dependent on the expression of membrane-associated viral antigenicity. As soon as 5 d after RadLV inoculation, and during the entire leukemogenic process, suppressor T cells are detectable in the spleen that are capable of specifically abrogating generation of syngeneic anti-tumor cytotoxic cells in vitro. Mice exposed to fractionated x irradiation do not develop suppressor cells and their splenocytes may be stimulated in vitro to generate cytotoxicity toward RadLV-induced leukemias. These findings suggest that although RadLV has been isolated from radiation-induced leukemias, x-ray- and RadLV-induced leukemogenesis do not seem to involve a common viral etiology, and that induction of suppressor cells during RadLV leukemogenesis may be essential for tumor progression.

Infant mouse as a sensitive bioassay system for carcinogenicity of N-nitroso compounds.

Abstract: Five groups of 48, C57BLxC3H F1, male mice, 15 days old, were administered NDEA intraperitoneally at the following levels: 0.0 (saline only), 0.625, 1.25, 2.50 and 5.00 microgram per g body weight, in a single dose. Groups of eight mice from each of the dose levels were killed at 40, 50, 60, 70, 80 and 90 weeks of age. The nodular liver lesions were classified s (a) focal areas of non-specific cellular changes, (b) hyperplastic nodules, (c) hepatocellular adenomas and (d) hepatocellular carcinomas. Regardless of the dose, all the animals developed hepatocellular carcinomas. The average latent periods, however, were inversely proportional to dose, being 66 weeks with the lowest dose and 44 weeks with the highest dose. The multiplicity and the average weight of the early nodular lesions (50 weeks) was directly related to th NDEA dose. Thus the higher multiplicity was associated with faster emergence of hepatocellular carcinomas. The high susceptibility to hepatocellular carcinogenesis, in association with the short latency, makes the infant mouse a sensitive bioassay system for assessing the carcinogenic potential of N-nitroso compounds.

Hepatic drug metabolizing enzyme activity and tumorigenesis in mice following perinatal exposure to benzo(a)pyrene.

Abstract: It is known that in utero exposure to polycyclic aromatic hydrocarbon carcinogens (PAH) results in a high incidence of pulmonary and other tumors in the offspring, usually after a long latency period. The present study tested the hypothesis that perinatal exposure to benzo(a)pyrene (BP) produces alterations in microsomal mixed-function oxidase (MFO) activity that modify the response to postnatal challenge with a PAH in such a way as to render the offspring to be more susceptible to tumorigenesis, and to determine whether the gestational age at which BP exposure occurred was a determinant of the proposed alterations. Swiss mice were treated with BP on day 3, 8, 10, 12, 14, or 18 of gestation. Subgroups from each gestational-age treatment group and appropriate controls were studied for enzyme activity or were challenged with 3-methylcholanthrene (3-MC) when three months old. Female offspring of BP-treated mice had significantly elevated hepatic microsomal aminopyrine demethylase activity; cytochrome P-450 concentrations were significantly lower in male offspring. The most striking finding was the increased susceptibility to tumorigenesis of female offspring. Latency for local tumors was decreased by two weeks; tumors were more frequent and grew more rapidly. Significantly more female offspring had both local and pulmonary lesions, and the total number of pulmonary adenomas was also significantly higher. Further studies are required to determine if the developmental alterations in hepatic (MFO) activity resulting from perinatal exposure to PAH relate in a causative way to the enhanced susceptibility of female offspring to tumorigenesis.

Tumorigenesis by benzo(a)pyrene administered intracolonically.

Abstract: Benzo(a)pyrene (BP) was administered to Swiss mice in 1 and 10 weekly intracolonic instillations at 200 μ g/g body weight. The single administration of BP induced a statistically sig

Toxicological significance of liver hypertrophy produced by inducers of drug-metabolizing enzymes.

Abstract: Changes in enzyme activity due to induction by chemicals is an important property that can determine the type of response seen in tissues exposed to environmental chemicals Two major types of response, acute irreversible liver cell injury or death (necrosis) and long-term cancer induction, are discussed in terms of their modulation by enzyme induction Most commonly, enzyme induction leads to a more severe toxic response by the liver, and to more cell death However, inducers may have a protective effect, especially in carcinogenesis, when they most frequently protect against cancer induction if used early in the process There is a discrepancy between this observation and the increase in mutagenic activity of liver preparations observed after induction However, when enzyme induction occurs at a later stage, after initiation, it often accelerates or promotes cancer induction Also, new cell populations constantly observed during liver carcinogenesis are composed of very hypertrophic hepatocytes containing a large amount of smooth endoplasmic reticulum This is associated with a radical change in enzyme activities in the reticulum, which may account in part for the characteristic resistance exhibited by initiated cells to hepatotoxins and carcinogens The resistance is considered to be an important property that may play a key role in the development of cancer under some circumstances

Low molecular weight peptidic fraction in the chromatin from normal and cancer cells: Control of transcription

Abstract: DNA isolated from cell nuclei by intensive deproteinization with chloroform/isoamyl alcohol and phenol extractions contains a low molecular weight peptidic fraction in a quantity of about 20 μg/mg DNA. These peptides were characterized by chromatography on CM-Sephadex, Sephadex G-25, high performance liquid chromatography on μBondapak C18 and amino acid composition. The peptides control transcription in a reconstituted cell-free system with prokaryotic and eukaryotic RNA polymerase and stabilize the structure of double stranded DNA, while increasing its melting point. Their level is markedly decreased (by about 40%) in DNA prepared from tumor cells as compared to normal cell DNA. Transcriptional studies showed only a slightly increased template activity of DNA extracted at pH 9.5 versus DNA extracted at pH 6.0 for DNA preparations from tumor cells. However, there was a marked increase in template activity for DNA preparations treated at pH 9.5 from normal cells-232%, 124%, 97% and 78% for rat liver, mouse liver, mouse thymus and fibroblast L-929 cells, respectively. Also there was no difference in the melting point between these two preparations of DNA from tumor cells; normal cell DNA preparations showed increased melting point of preparations treated at pH 6.5. The data obtained indicate that the loss of low molecular weight peptides from tumor DNA during carcinogenesis is responsible for uncontrolled gene expression observed in cancer.

Autoradiographic Demonstration of DNA Repair Synthesis in Rat Tracheal Epithelium Treated with Chemical Carcinogens in Vitro

Abstract: A system in which tracheal organ culture of inbred F344 rats was used in combination with autoradiography was developed for quantitative measurement of DNA repair synthesis in tracheal epithelial cells. Small sections of trachea in short-term organ culture were treated with various carcinogens plus [methyl-3H]thymidine. Significant numbers of grains, indicating DNA repair, were detected on the nuclei of epithelial cells of tracheal sections treated with carcinogens, and the numbers were proportioned to the concentrations of the carcinogens. The nuclear [methyl-3H]thymidine labeling index (S-phase index) was approximately 0.02, and this did not interfere appreciably with quantitative grain counting. This autoradiographic method is suitable for quantitative measurement of DNA repair synthesis in epithelial cells of the trachea in conditions mimicking those in vivo. These results suggest the potential use of this system for studies on the mechanism of carcinogenesis at a cellular level in keeping with recent biochemical studies on metabolism of carcinogens in respiratory organs in culture. The system may also be useful for screening environmental chemicals suspected of damaging DNA of the respiratory organs in relation to lung carcinogenesis.

Presensitization of human cells with extrinsic signals to induced chemical carcinogenesis

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