Establishment of epithelial cell lines from adult mouse regenerating liver

A simple technique for developing epithelial cell lines from regenerating mouse liver has been described. Twenty-one epithelial cell lines have been developed and can be divided into four groups according to their morphology. All these near diploid cell lines have the capacity to metabolize diverse classes of chemical carcinogens (3-methylcholanthrene, 2-acetylaminofluorene, dimethylnitrosamine and aflatoxin B1) to cytotoxic metabolites. It is not yet possible to determine which ones of these cell lines originated from hepatocytes. Studies are in progress to further characterize and to use these cell lines as lethally irradiated feeder layers for cell-mediated activation of various classes of chemical carcinogens and mutagens with C3H/10T1/2 mouse embryo fibroblasts as indicator cells.     

Metabolism of Benzo(a)pyrene by Human Epithelial and Fibroblastic Cells: Metabolite Patterns and DNA Adduct Formation

We demonstrate in cell culture that mammary epithelial cells from normal human breast specimens metabolize benzo(a)pyrene (BaP) and form adducts with the bases of their DNA more readily and at lower concentrations of BaP than do fibroblasts from the same specimens. BaP metabolism and adduct formation was determined in the same incubations with epithelial cells grown out in early passage from each of three specimens and with fibroblasts from one of these specimens. The metabolite pattern of the epithelial cells was indicative of preferential formation of 7, 8-dihydrodiol-9, 10-dihydroepoxybenzo(a)pyrene the ultimate carcinogen. In contrast, fibroblasts formed mainly mono- and dihydroxide derivatives of BaP. The metabolite pattern from epithelial cells was compatible with the ease in which adducts between DNA and the diolepoxide of benzo(a)pyrene were formed. These results provide evidence that chemical carcinogens should be considered as possible factors in the induction of breast cancer in women.     

The effect of radiation in combination with carcinogens on the growth of normal urothelium in explant culture

Summary Radiation is known to be carcinogenic to humans but attempts to demonstrate the process using human tissue culture models have met with little success. In the present study explants were established from urothelium and exposed to radiation and a range of chemical carcinogens, suspected promotor or metabolic agents. The resulting outgrowth was monitored for growth rate, proliferating epithelial fraction and development and differentiation of endothelial cells in culture. The results indicate that enhanced growth of epithelial cells can be seen when cultures are irradiated in the presence of various nitrosamines, benzo(a)pyrene or aniline. Radiation alone reduced the overall growth area measured but several proliferative foci developed on the resulting outgrowth. Their ultrastructural appearance reveals that they carry severe mitochondrial damage and exposure of treated cultures to metabolic inhibitors confirms that their respiration is defective. Endothelial cells proliferated over the surface of the epithelial monolayer and both the number and the degree of differentiation of the endothelial cells increased with increasing dose up to 10 Gy. While the cultures are not immortalised by the treatment, it appears that the epithelial cells have an extended lifespan (division capacity) and that a subpopulation has undergone a number of premalignant changes. Changes in endothelial cell proliferation also occur.     

Inflammation as the primary aetiological agent of human prostate cancer: A stem cell connection?

Inflammation has been implicated for some time as a potential aetiological agent in human prostate cancer. Viral and bacterial infections or even chemical carcinogens such as those found in cooked meat have been proposed as the inflammatory stimuli, but the mechanism of cancer induction is unknown. Recent information about gene expression patterns in normal and malignant epithelial stem cells from human prostate provides a new hypothesis for inflammation-induced carcinogenesis. The hypothesis states that in the stem cells located in the basal cell compartment of the prostate, activated prostate epithelial stem cells acquire a survival advantage, by expressing one of more of the same cytokines such as IL6. The establishment of one or more autocrine signalling loops results in an expansion of these cells in the absence of inflammation, as a potential first stage in the development of the tumour.     

Induction of sister-chromatid exchanges by procarcinogens in metabolically competent Chinese hamster epithelial liver cells

An epithelial cell strain has been established from the livers of male Chinese hamsters (CHEL cells). These cells, which proliferate in culture and retain their metabolic enzymatic activities during several subcultures, were used in a sister-chromatid exchange assay to evaluate the effectiveness of polycyclic aromatic hydrocarbons (PAHs), aflatoxin B1 (AFB1) and cyclophosphamide (CP). The results obtained demonstrate that CHEL cells are metabolically competent to activate different classes of procarcinogens into biologically active metabolites. Moreover, they showed a selective capacity to discriminate chemical carcinogens from noncarcinogens. Thus, the CHEL cell system appears to be a promising alternative to the short-term tests that include cell-free rodent liver homogenate to evaluate new promutagens and/or procarcinogens.     

Mismatch repair processing of carcinogen-DNA adducts triggers apoptosis

The DNA mismatch repair pathway is well known for its role in correcting biosynthetic errors of DNA replication. We report here a novel role for mismatch repair in signaling programmed cell death in response to DNA damage induced by chemical carcinogens. Cells proficient in mismatch repair were highly sensitive to the cytotoxic effects of chemical carcinogens, while cells defective in either human MutS or MutL homologs were relatively insensitive. Since wild-type cells but not mutant cells underwent apoptosis upon treatment with chemical carcinogens, the apoptotic response is dependent on a functional mismatch repair system. By analyzing p53 expression in several pairs of cell lines, we found that the mismatch repair-dependent apoptotic response was mediated through both p53-dependent and p53-independent pathways. In vitro biochemical studies demonstrated that the human mismatch recognition proteins hMutSalpha and hMutSbeta efficiently recognized DNA damage induced by chemical carcinogens, suggesting a direct participation of mismatch repair proteins in mediating the apoptotic response. Taken together, these studies further elucidate the mechanism by which mismatch repair deficiency predisposes to cancer, i.e., the deficiency not only causes a failure to repair mismatches generated during DNA metabolism but also fails to direct damaged and mutation-prone cells to commit suicide.     

Enhanced proliferation of cells from human tissue explants following irradiation in the presence of environmental carcinogens

Summary A method which allows growth of normal human tissue to be studied in vitro is used to investigate possible interactive effects of radiation and environmentally important carcinogens on oesophageal and urothelial cell growth. Carcinogens chosen were selected for their known or suspected effect on the oesophageal mucosa or urothelium in vivo. The results indicate that with carcinogens alone concentrations can be identified that result in increased proliferation of cells. With radiation alone inhibition of cell proliferation occurs at all dose points examined. However, at precise combinations of radiation and carcinogen, greatly enhanced cell proliferation could be detected, suggesting a synergistic interaction between the two agents. The results may have implications for the design and interpretation of experiments aimed at elucidating early or premalignant changes in epithelial tissues and may indicate hitherto unsuspected interactions between radiation and environmentally important carcinogens.     

Tissue-specific mutagenesis by N-butyl-N-(4-hydroxybutyl)nitrosamine as the basis for urothelial carcinogenesis

Bladder cancer is one of the few cancers that have been linked to carcinogens in the environment and tobacco smoke. Of the carcinogens tested in mouse chemical carcinogenesis models, N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) is one that reproducibly causes high-grade, invasive cancers in the urinary bladder, but not in any other tissues. However, the basis for such a high-level tissue-specificity has not been explored. Using mutagenesis in lacI (Big Blue?) mice, we show here that BBN is a potent mutagen and it causes high-level of mutagenesis specifically in the epithelial cells (urothelial) of the urinary bladder. After a 2-6-week treatment of 0.05% BBN in the drinking water, mutagenesis in urothelial cells of male and female mice was about two orders of magnitude greater than the spontaneous mutation background. In contrast, mutagenesis in smooth muscle cells of the urinary bladder was about five times lower than in urothelial tissue. No appreciable increase in mutagenesis was observed in kidney, ureter, liver or forestomach. In lacI (Big Blue?) rats, BBN mutagenesis was also elevated in urothelial cells, albeit not nearly as profoundly as in mice. This provides a potential explanation as to why rats are less prone than mice to the formation of aggressive form of bladder cancer induced by BBN. Our results suggest that the propensity to BBN-triggered mutagenesis of urothelial cells underlies its heightened susceptibility to this carcinogen and that mutagenesis induced by BBN represents a novel model for initiation of bladder carcinogenesis.     

v-ras genes from Harvey and BALB murine sarcoma viruses can act as initiators of two-stage mouse skin carcinogenesis

Activated Harvey murine sarcoma virus ras genes were introduced into epidermal cells in vivo by direct application of retroviruses to mouse skin. Subsequent treatment with the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) induced benign papillomas, some of which progressed to invasive carcinomas. Initiation with virus was irreversible for at least 4 months, since TPA treatment after this latency period produced papillomas within 4 weeks. Analysis of viral integration sites showed that carcinomas are clonal in origin. Both papillomas and carcinomas express virus-specific ras mRNA and the viral form of ras P21 protein. The results show that activated ras genes can replace chemical carcinogens in initiation of mouse skin carcinogenesis. This system presents a novel approach to in vivo analysis of the biological role of oncogenes in epithelial tumorigenesis.     

Stem cell characteristics of transplanted rat mammary clonogens

Rat mammary glands contain a subpopulation of clonogenic epithelial cells with large proliferation and differentiation potentials. When transplanted, the clonogens in monodispersed rat mammary epithelial cell suspensions give rise to either alveolar units (AUs) or ductal units (DUs) depending on the nature of the hormonal milieu in the graft recipient. Clonogens are also the primary cells of origin of mammary cancer following exposure to ionizing radiation or chemical carcinogens. Given the other stem cell characteristics of mammary clonogens, it would be expected that the primary AUs and DUs to which they give rise when grafted and hormonally stimulated (a) would be derived from the same clonogenic cell subpopulation, (b) would contain all of the functionally differentiated cell types of homologous parts of comparably stimulated mammary glands in situ, and (c) would also contain clonogen subpopulations capable when subtransplanted of giving rise to secondary AUs and DUs of similar cell composition. The current experiments were designed to test these expectations. The data are discussed in the context of results of previous studies with this and other experimental models. The results further support the conclusion that rat mammary clonogens are multipotent mammary stem cells.     

A diploid epithelial cell line from normal adult rat liver with phenotypic properties of 'oval' cells

A diploid epithelial cell line (termed WB-F344) was isolated from the liver of an adult male Fischer-344 rat and the phenotypic characteristics of the cells were studied. These cells measure approximately two-fifths the volume of freshly isolated hepatocytes. They are histochemically negative for glucose-6-phosphatase and weakly positive for gamma-glutamyl transpeptidase. They produce extensive intercellular reticulin fibers which stain immunocytochemically for fibronectin, and they synthesize both alpha-fetoprotein and albumin, but they do not accumulate glycogen particles. Ultrastructurally, they are polygonal cells with numerous intercellular desmosomes and nexus junctions, and they are partially surrounded by basement membrane-like material. Cytoplasmic organelles include few, but sometimes dilated profiles of rough endoplasmic reticulum, lysosomes, abundant free ribosomes, sparse smooth endoplasmic reticulum and Golgi membranes, microbodies, and small, pleomorphic mitochondria. They express A and C isozymes of aldolase, K isozyme of pyruvate kinase, LDH2 to LDH5 isozymes of lactate dehydrogenase, and 'fetal liver'-type alkaline phosphatase isozyme. When compared with the phenotypes of isolated and purified normal hepatocytes, biliary epithelial (ductular) cells and 'oval' cells isolated from livers treated with chemical carcinogens, the phenotypic properties of the liver epithelial cell line in culture most resemble those of the 'oval' cells.     

Chemical interactions with herpes simplex type 2 virus: Enhancement of transformation by hydrazine and 1,2-dimethylhydrazine

Quantitative assays for the morphological transformation of 3T3 Swiss mouse cells by herpes simplex type 2 virus (HSV-2) were employed to examine the effect on cell transformation of chemical carcinogens and suspected carcinogens. Exposure of the cells to the chemical compound, followed by virus infection, resulted in enhancement of transformation when compared to that observed with chemical or virus alone. Enhancement occurred in tests utilizing either UV light-inactivated HSV-2 (strain 333) or a temperature-sensitive (ts) mutant of HSV-2 [A₈(293)]. A series of seven ts-mutants were tested and exhibited varying degrees of transformation. Enhancement of transformation occurred in cells treated with hydrazine (HZ) and 1,2-dimethylhydrazine (SDMH). No enhancement occurred when cells were treated with monomethylhydrazine, 1,1-dimethylhydrazine and the jet fuels JP-5, JP-10, RJ-4 and RJ-5. A strong time dependence after treatment was demonstrated with some enhancement seen at 6 h after chemical treatment but the greatest enhancement appeared when virus infection began after 24 h of chemical exposure.     

A role for fibroblasts in mediating the effects of tobacco-induced epithelial cell growth and invasion

Cigarette smoke and smokeless tobacco extracts contain multiple carcinogenic compounds, but little is known about the mechanisms by which tumors develop and progress upon chronic exposure to carcinogens such as those present in tobacco products. Here, we examine the effects of smokeless tobacco extracts on human oral fibroblasts. We show that smokeless tobacco extracts elevated the levels of intracellular reactive oxygen, oxidative DNA damage, and DNA double-strand breaks in a dose-dependent manner. Extended exposure to extracts induced fibroblasts to undergo a senescence-like growth arrest, with striking accompanying changes in the secretory phenotype. Using cocultures of smokeless tobacco extracts-exposed fibroblasts and immortalized but nontumorigenic keratinocytes, we further show that factors secreted by extracts-modified fibroblasts increase the proliferation and invasiveness of partially transformed epithelial cells, but not their normal counterparts. In addition, smokeless tobacco extracts-exposed fibroblasts caused partially transformed keratinocytes to lose the expression of E-cadherin and ZO-1, as well as involucrin, changes that are indicative of compromised epithelial function and commonly associated with malignant progression. Together, our results suggest that fibroblasts may contribute to tumorigenesis indirectly by increasing epithelial cell aggressiveness. Thus, tobacco may not only initiate mutagenic changes in epithelial cells but also promote the growth and invasion of mutant cells by creating a procarcinogenic stromal environment.     

Various Levels of DNA Repair Synthesis in Xeroderma Pigmentosum Cells exposed to the Carcinogens N-Hydroxy and N-Acetoxy-2-acetyl-aminofluorene

IN assessing environmental health hazards, the question has arisen of whether “safe”, “tolerable” or “permissible” levels of carcinogens, mutagens or teratogens can be derived by extrapolation of bioassays using rodents exposed for various periods to very high concentrations of chemicals or using cultured mammalian cell lines. Variations in susceptibility are only rarely taken into account, if at all and doses which seem to be harmless to the average person may be harmful to susceptible people. The reduced capacity to repair ultraviolet-induced DNA lesions in xeroderma pigmentosum (XP) cells may exemplify a mechanism leading to an elevated neoplastic transformation rate in man^1–4. The question arises as to whether cells with deficient repair synthesis respond to chemical carcinogens in the same manner as cells with adequate repair systems. We report here the levels of DNA repair synthesis in XP cells of five patients exposed to the carcinogenic^5,6 and mutagenic⁷ compounds N-acetoxy or N-hydroxy-2-acetyl-aminofluorene, which are ultimate and proximate carcinogenic forms of 2-acetylaminofluorene (AAF). We were particularly interested in comparing the different levels of DNA repair synthesis following ultraviolet irradiation with those following treatment with chemical carcinogens.     

Enhancement of the morphological transformation of Syrian hamster embryo (SHE) cells by reducing incubation time of the target cells

Syrian hamster embryo (SHE) cell transformation has been used for many years to study chemical carcinogenesis in vitro. It has been shown that this assay is probably the most predictive short-term test system for identifying rodent carcinogens. Although most of the operational difficulties encountered in the early stage of application of this assay have been overcome by culturing the SHE cells under slightly acidic conditions (pH 6.7), a relatively low level of induction of morphological transformation (MT) by known carcinogens still occurs for many cell isolates. In order to improve the response of this assay system to known carcinogens, the effect of incubation time of target SHE cells on the frequency of morphological transformation induced by benzo(a)pyrene (BaP) was investigated. It was shown that the morphological transformation frequency induced by BaP increased significantly (1.4-2.5-fold) when the incubation time of target cells was reduced from the usual 24h to less than 6h prior to seeding onto feeder layers. This improvement in sensitivity was consistent for different cell isolates. In addition, the enhanced response appeared to be a property of carcinogens because treatment with two non-carcinogens, l-ascorbic acid and 4-nitro-o-phenylenediamine, did not induce significant increases in the transformation frequency under the shortened incubation period for target cells. These results suggest that the response of the SHE cell transformation assay may be improved by optimizing the incubation time of the target SHE cells. In addition, the results of the present study provide further evidence to support the idea that morphological transformation of SHE cells results from a block of cellular differentiation of stem or stem-like cells.     

Quantitation of cell proliferation,colony formation,and carcinogen induced cytotoxicity of rat tracheal epithelial cells grown in culture on 3T3 feeder layers

Summary A cell culture system is described for the growth of rat tracheal epithelial (RTE) cells at clonal density. The system uses normal, early passage RTE cells grown on feeder layers of lethally irradiated 3T3 cells. The RTE cells have a high colony forming efficiency (5 to 10%) in culture, can be passaged up to 5 times, and are capable of more than 20 cumulative doublings per colony forming cell. The epithelial nature of the cells was confirmed by cell and colony morphology, immunoperoxidase staining of intracellular keratin, and cellular ultrastructural studies. The cytotoxic response of RTE cells to a variety of carcinogens, including a direct acting chemical carcinogen, a physical carcinogen, and a series of polycyclic aromatic hydrocarbons, was quantitated. A linear decrease in the logarithm of survival was observed with increasing doses ofN-methyl-N′-nitro-N-nitrosoguanidine (MNNG), γ-irradiation, 7,12-dimethylbenz(a)anthracene, and a diol-epoxide of benzo(a)pyrene. No toxicity was observed after treatment with benzo(a)pyrene or 3-methylcholanthrene over the concentration range examined. In contrast, phorbol ester tumor promoters stimulated cell growth markedly. Based on these and other studies, the RTE cell culture system represents a model system that will be useful for quantitative studies of epithelial cell growth, differentiation, and carcinogenesis.     

Heat shock and arsenite increase expression of the multidrug resistance (MDR1) gene in human renal carcinoma cells

The multidrug transporter, initially identified as a multidrug efflux pump responsible for resistance of cultured cells to natural product cytotoxic drugs, is normally expressed on the apical membranes of excretory epithelial cells in the liver, kidney, and intestine. This localization suggests that the multidrug transporter may have a normal physiological role in transporting cytotoxic compounds or metabolites. In the liver, hepatectomy or treatment with chemical carcinogens increases expression of the MDR1 gene which encodes the multidrug transporter. To evaluate conditions which increase MDR1 gene expression, we have investigated the induction of the MDR1 gene by physical and chemical environmental insults in the renal adenocarcinoma cell line HTB-46. There are two strong heat shock consensus elements in the major MDR1 gene promoter. Exposure of HTB-46 cells to heat shock, sodium arsenite, or cadmium chloride led to a 7- to 8-fold increase in MDR1 mRNA levels. MDR1 RNA levels did not change following glucose starvation or treatment with 2-deoxyglucose and the calcium ionophore A23187, conditions which are known to activate the expression of another family of stress proteins, the glucose-regulated proteins. The levels of the multidrug transporter, P-glycoprotein, as measured by immunoprecipitation, were also increased after heat shock and sodium arsenite treatment. This increase in the level of the multidrug transporter in HTB-46 cells correlated with a transient increase in resistance to vinblastine following heat shock and arsenite treatment. These results suggest that the MDR1 gene is regulatable by environmental stress.     

The Ty1 transposition assay: a new short-term test for detection of carcinogens

An assay based on induction by carcinogens of Ty1 transposition in Saccharomyces cerevisiae is proposed. A tester strain was developed that contains a marked Ty1 element, which allows following the transposition in the genome as a whole and a mutation, which increases cellular permeability. Hypersensitivity to chemical agents, higher cell wall porosity and transformability with plasmid DNA evidenced an enhanced cellular permeability of the tester cells. The increased permeability resulted in higher sensitivity to carcinogens. The treatment with different laboratory carcinogens induced Ty1 transposition rates in the tester strain by a factor of 10 to 20, compared to the controls. The induction is not stress-generated by the cytotoxicity of carcinogens, since treatment with NaN3 at concentrations killing 50% of the cells did not increase the transposition rate. The increase of Ty1 transposition in tester cells is specific for active carcinogens and a positive response with procarcinogens was obtained only in presence of S9 mix. The Ty1 transposition test responded positively to a number of Ames-test or DEL-test negative carcinogens. The positive response of Ty1 test was statistically significant and verified in kinetics and concentration-dependent experiments. It is concluded that the Ty1 transposition test can be used, in addition to the Ames assay, as a short-term test for detection of carcinogens.