Search

Multi-stage carcinogenesis (initiation-promotion) was first demonstrated in mouse skin. The first stage, initiation, is accomplished by a low dose of carcinogen that causes no tumours. Promotion by repeated treatment of initiated mice with certain non-carcinogenic hyperplastic agents results in the rapid production of numerous benign papillomas, a few of which progress to squamous cell carcinomas. Although this models system produces mostly benign tumours, many of the concepts concerning carcinogenesis in epithelial tissues have been derived from mouse skin studies. The permanent change in growth potential accomplished by tumour initiators is generally considered to be a mutagenic event; cell selection and clonal expansion of initiated cells may be involved in promotion. In initiation-promotion experiments, more than 90% of the squamous cell carcinomas develop from papillomas, but the conversion rate is low. The factors necessary for this conversion of benign to malignant tumours have not been defined but tumour promoters have been assumed to be involved. However, we report here that the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) is ineffective in the conversion of papillomas to carcinomas whereas three initiators, urethane, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and 4-nitroquinoline-N-oxide (4-NQO) are effective. This suggests that malignant conversion may result from a further genetic change in papilloma cells and that the ineffectiveness of TPA may be due to its inactivity as a mutagen.

The intensities of immunofluorescence in nuclei stained by an antiserum specific for the DNA adduct N-deoxyguanosin(8-yl)aminofluorene (dG-8-AF), were quantified by microfluorometry in frozen liver sections from male Fischer rats fed 2-acetylaminofluorene (AAF). Results of previous studies demonstrated that dG-8-AF is the predominant adduct (80-100%) formed in livers of rats fed AAF continuously, and that nuclei of hepatocytes and bile duct epithelial cells in rats fed AAF exhibit an adduct-specific immunofluorescence. In the present investigation, nuclear staining for dG-8-AF was quantified by microfluorometry in liver sections from male Fischer rats fed 0.02% AAF continuously for 2, 4, 8, 12, 16, 20, and 28 days. Microfluorometric determinations of the intensities of nuclear immunofluorescence staining within periportal, midzonal, and centrilobular hepatocytes and bile duct epithelial cells revealed that levels of the dG-8-AF adduct increased in these cells during AAF feeding, reaching a plateau by 12 days. However, significant differences were detected in dG-8-AF levels within cells of each lobular area. Nuclei of periportal hepatocytes exhibited the most intense immunofluorescence, nuclei of centrilobular hepatocytes and bile duct epithelial cells emitted the least intense fluorescence, and nuclei of midzonal hepatocytes exhibited an intermediate fluorescence intensity. Quantitation of whole-liver levels of the dG-8-AF adduct by RIA, after extraction of DNA, also revealed that adduct accumulation reached a plateau by 12 days of AAF feeding. Thus, similar profiles of adduct accumulation were obtained by microfluorometric analysis of immunofluorescence staining within frozen liver sections, and by RIA analysis of DNA extracted from whole livers. The periportal concentration of DNA adducts in livers of rats continuously fed a carcinogenic dose of AAF may be an important early event in AAF-induced liver tumorigenesis.

SENCAR mice are extremely susceptible to two-stage skin carcinogenesis, while BALB/c mice are relatively resistant. Skin grafts to BALB/c nude mice were performed with full-thickness skin from SENCAR and BALB/c donors, and tumor formation was monitored in grafted skin, surrounding host skin, and intact SENCAR, BALB/c and nude mice. Initiation was accomplished by exposure to 20 micrograms dimethylbenz(a)anthracene and promotion by repeated exposure to 12-O-tetradecanoylphorbol-13-acetate. SENCAR skin retained a high sensitivity to carcinogenesis when grafted to nude hosts, whereas BALB/c skin remained resistant. The donor type did not influence the tumor yield in surrounding nude host skin. The rate of tumor regression was not altered in SENCAR skin grafts on nude mice relative to intact SENCAR skin. These results indicate that the unusual sensitivity of SENCAR epidermis to chemical carcinogenesis is not due to altered systemic factors but is a property of the skin itself.

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.

Studies of the mutagenic action required for specific chemicals to produce benign or malignant tumours suggest that in mouse skin at least two genetic events occur before carcinoma formation. The isolation of an activated form of the c-rasH gene from skin papillomas has provided evidence that this gene may be a target for the first mutation, which could constitute the initiating mutation in skin carcinogenesis. In vitro studies indicate that the v-rasH gene of Harvey murine sarcoma virus (Ha-MSV), a replication-defective transforming retrovirus, could impart a conditional initiated phenotype on cultured keratinocytes by blocking their ability to differentiate terminally and arresting them in a late basal cell stage of maturation. We now show that when the Ha-MSV v-rasH gene is introduced into cultured keratinocytes by a defective retroviral vector, skin grafts constructed with cells carrying the mutated ras oncogene produce papillomas on athymic nude mouse recipients. Furthermore, the expression of the exogenous oncogene seems to be regulated at the transcriptional level in the differentiated portions of the benign tumour.

Papillomas induced by standard initiation-promotion protocols progress to carcinomas at a low frequency. Experimental protocols were developed to elicit papillomas with a higher probability of malignant conversion. SENCAR mice initiated by 7,12-dimethylbenz[a]anthracene were promoted by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) for 5, 10, 20 or 40 weeks. With promotion for 10 weeks or more, a peak of papilloma incidence at 16-20 weeks was followed by a 35-40% decrease within 3 months. A much lower papilloma response was seen in mice promoted for 5 weeks, but these papillomas persisted. The yield of malignant tumors was similar in all four groups, with 20-25 carcinomas per group of 30 mice. Thus, the papillomas induced by the first few TPA treatments are much more likely to progress to carcinomas than those which appear later. In a separate study, initiated Charles River CD-1 mice were promoted with TPA for either 12 or 52 weeks. Acetone solvent treatment was begun at Week 13 in the group treated 12 weeks with TPA. At Week 16, the papilloma incidence was identical in the two groups of mice. However, by Week 28, the papilloma yield in the continuous TPA group had increased and was twice that of the acetone group, in which papillomas had regressed. The first carcinoma arose 14 weeks earlier with continuous TPA, but the final number of carcinomas per group of 40 mice was 17 with TPA and 20 with acetone. Neither the increase in papillomas in TPA-treated mice nor the regression of papillomas after cessation of promotion with TPA affected the final carcinoma yield. This result suggests that TPA-dependent papillomas are very unlikely to progress to carcinomas. In a third experiment, promotion of initiated SENCAR mice with mezerein resulted in a small number of papillomas which had a much higher probability of progression to carcinomas than the large number of papillomas promoted by TPA. The ability to induce papillomas promoted by TPA. The ability to induce papillomas with a known probability of conversion to carcinomas will facilitate the identification of markers associated with malignant progression.

Histological studies using paired immunofluorescence staining and peroxidase-anti-peroxidase staining were performed on sections of rat livers with an antiserum specific for the 2-acetylaminofluorene (AAF)-DNA adduct N-deoxyguanosin-(8-yl)-aminofluorene (dG-8-AF). This is the predominant adduct in rat liver DNA at 5 (80%) and 28 (100%) days of AAF feeding. Nuclear staining was observed in livers of male Fischer rats fed 0.02% AAF for these time periods, and was not present in livers of animals fed control diet or detected when specific antiserum, first absorbed with the immunogen adduct, was utilized. In addition, nuclear staining was unchanged after incubation with RNase and abolished after incubation with DNase. Adducts were not readily detectable when whole-liver adduct concentrations were less than an average of 10(5) adducts per cell (30-50 fmol/micrograms DNA). The overall pattern of adduct distribution in livers of AAF-fed animals was distinctly non-uniform. A predominance of nuclear staining was found in the periportal areas by both immunofluorescence and immunoperoxidase procedures. In contrast, staining was very weak in the centrilobular areas. When animals were fed AAF for 28 days and control diet subsequently for 7, 14, 21 or 28 days, the overall intensity of the immunohistochemical staining decreased with time on control diet. However, the pattern of localization remained the same as in livers of rats fed AAF for 28 days, with the predominance of adducts being in the periportal areas. In male rats fed 0.02% AAF for 8 weeks, foci positive for gamma-glutamyltranspeptidase (GGT) became apparent, and the nuclei in these areas showed no immunofluorescence, indicating the absence of detectable levels of the dG-8-AF adduct. Twenty adduct-negative areas in the median lobes of three rat livers were positive for GGT, which suggests that loss of ability to form adducts in these regions occurs concomitantly with early phenotypic changes.

The pathology of 60 aged female SENCAR mice used as acetone controls in skin painting studies was studied. Fifty percent of the mice survived past 96 weeks of age. The major contributing causes of death identified in 42 mice were glomerulonephritis (8 mice), histiocytic sarcoma (7 mice), and other tumors (8 mice). Glomerulonephritis was found in the majority of mice and was associated with thymic hyperplasia, focal vasculitis, and lymphoid hyperplasia. Necropsy of 58 mice surviving past 50 weeks of age revealed that 41 had an average of 1.36 tumors per mouse. The most common tumors included histiocytic sarcoma (13 mice), pulmonary adenoma or adenocarcinoma (11 mice), mammary tumors (11 mice), follicular center cell lymphoma (4 mice), and hepatocellular adenoma (4 mice). The 13 histiocytic sarcomas appeared to arise in the uterus and metastasized to liver (9 mice), lung (4 mice), kidney (3 mice), and other tissues. Lung tumors were of the solid and papillary types, and tumor cells frequently contained surfactant apoprotein (SAP) but did not contain Clara cell antigens, suggesting their origin from alveolar Type II cells. A variety of nonneoplastic lesions, similar to those observed in other mouse strains, were seen in other tissues of these mice. Amyloid-like material was seen only in nasal turbinates and thyroid gland. In a group of 28 mice exposed to 12-O-tetradecanoylphorbol-13-acetate (TPA) for up to 88 weeks, as a control for other treatment groups, 7 (25%) had papillomas and 5 (17.8%) had squamous cell carcinomas of the skin at necropsy, although many other induced papillomas regressed during the study.

Di(2-ethylhexyl)phthalate (DEHP), a confirmed promoter of hepatocellular carcinogenesis in mice, was studied for tumor-promoting activity on the skin of SENCAR mice in vivo and on JB6 mouse epidermal cells in vitro. DEHP enhanced transformation to anchorage-independent growth of promotable JB6 clonal lines, C141, C121, and R219. DEHP induced colony formation in concentrations from 500 p.p.m. to 20 000 p.p.m./ml culture medium with maximum induction occurring at 10 000 p.p.m./ml. DEHP was inactive as a complete promoter of skin carcinogenesis initiated by 7,12-dimethylbenz[a]anthracene (DMBA) in SENCAR mice. Like the plant-derived natural product mezerein, however, DEHP significantly enhanced skin carcinogenesis in SENCAR mice when initiation by DMBA was followed by short term applications (2x/week, 2 weeks) of 12-O-tetradecanoylphorbol-13-acetate prior to application of DEHP. A random sample of the tumors observed was processed and examined histologically. Most neoplasms were papillomas; a few were squamous cell carcinomas. The kinds of tumors seen were identical with those reported in two-stage carcinogenesis experiments in SENCAR mice in which phorbol ester tumor promoters have been employed. Thus, DEHP promotes transformation in JB6 mouse epidermal cells and acts as a second-stage promoter in mouse skin carcinogenesis. DEHP therefore has promoting capability for at least two distinct kinds of epithelium in mice.

The progression of papillomas to squamous cell carcinomas (malignant conversion) was studied in the skin of SENCAR and Charles River CD-1 mice, using a three-stage treatment protocol. After initiation with 7,12-dimethylbenz(a)anthracene (DMBA) (stage 1) and limited promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA) (stage II), papilloma-bearing mice were treated (stage III) with either tumor initiators, such as urethane, N-methyl-N'nitro-N-nitrosoguanidine (MNNG) or 4-nitroquinoline-n-oxide (R-NQO), the promoter TPA, or solvent (acetone). Similar final carcinoma yields were found in the mice treated in stage III with TPA or acetone, although carcinomas developed earlier in the TPA-treated mice. In contrast, treatment with tumor initiators in stage III increased both the rate of appearance and the final yield of carcinomas. Similar results were obtained in both SENCAR and CD-1 mice. A papilloma stage appears to be necessary for carcinoma development since elimination of TPA treatment in stage II greatly reduced the incidence of both papillomas and carcinomas in both stocks of mice. The heterogeneity of papillomas with regard to progression to carcinomas is demonstrated by the low rate of conversion of TPA-dependent papillomas and the high rate of conversion of persistent papillomas in CD-1 mice. The carcinomas that develop using the three-stage regimen vary in metastatic potential. In CD-1 mice, the frequency of metastases to lymph nodes were similar in groups treated in stage III with MNNG, urethane, 4-NQO, TPA, or acetone, but treatment with urethane substantially increased metastases to the lung.(ABSTRACT TRUNCATED AT 250 WORDS)

Retinoids have chemopreventive activity for epithelial tumors in a variety of systems, including the two-stage tumorigenesis system of mouse skin in which only the promotion stage is inhibited. We asked whether dietary vitamin A deficiency could affect the skin tumorigenic response, prior to major changes in body weight or general health of the animals. Two regimens were tested to induce vitamin A deficiency. SENCAR mice were either (a) fed a vitamin A-deficient diet from 4 or 9 weeks of age or (b) their mothers were fed the diet from the time of birth of the experimental animals which were then weaned on the same diet. The latter regimen produced typical symptoms of vitamin A deficiency in the offspring by Weeks 12-14 and all the mice died by Week 19; the former regimen permitted sufficient accumulation of retinol and its esters to sustain life for up to 45 and 75 weeks, respectively, in the majority of mice. For our experiments, vitamin A depletion was produced by placing the mothers on the deficient diet at birth of the experimental animals. A single topical dose of 20 micrograms of 7,12-dimethylbenz(a)anthracene (DMBA) was used as the initiator at 3 weeks of age and 1 to 2 micrograms of 12-O-tetradecanoylphorbol-13-acetate (TPA) once weekly as the tumor promoter for 10 weeks (from Week 4 through 13 of the experiment). Fifty-five % of mice (n = 40) on Purina laboratory chow (mean body weight, 31.4 g) developed skin tumors (2.58 per mouse) at 12 weeks, versus 2.5% (0.05 papillomas per mouse) of mice (n = 40) kept on the purified vitamin A-deficient diet (mean body weight, 30.3 g), a 98% decrease in tumor/mouse. Retinoic acid (RA) (1-3 micrograms/g diet) supplementation after Week 12 caused a rapid tumorigenic response in 95% of the mice by week 22. This tumor response occurred to a reduced extent in the absence of continued TPA treatment up to Week 13. Even though tumor incidence increased within 1 week of RA and 95% of the mice showed the tumorigenic response, the number of tumors per mouse was about 50% of that observed in mice maintained on standard Purina diet. This was confirmed in an experiment in which the mice were maintained for life either on Purina or on the RA (3 micrograms/g) containing purified diet, the latter being the control group for the effect of vitamin A deficiency on skin tumorigenesis.(ABSTRACT TRUNCATED AT 400 WORDS)

Rapid, synchronous and repeating cycles of marginal retinoic acid sufficiency and deficiency were produced in rats by following an established protocol. During cycles 11-15 (343-464 days after weaning), 17 of 24 rats (71%) developed tumor-like masses in the connective tissue surrounding the formative ends of one or both upper incisor teeth. The masses were composed of cords of odontogenic epithelium surrounded by a mantle of mesenchyme. Lakes of predentine, associated with foci of keratinized epithelium, were randomly distributed. On the basis of current understanding of odontogenesis, we propose that periodically disturbed differentiation of pulpal mesenchymal cells to dentin-secreting odontoblasts was pivotal to development of the masses. During deficiency, dentin secretion is impaired, the dentin wall perforates and with time the odontogenic epithelium and pulp herniate into the surrounding connective tissues. We propose that the incisal masses arose because the pulpal mesenchyme continued to grow and its secretion product, dentine, continued to be deposited (during periods of vitamin A sufficiency) in an ectopic site where functional attrition from mastication could not occur.

Methods to culture cells from papillomas induced by an initiation-promotion protocol in SENCAR mice were developed, and the resultant cell lines have been characterized. Using Eagle's medium with 0.05 mM Ca2+ conditioned by dermal fibroblasts and supplemented with 1 ng/ml epidermal growth factor (EGF) in culture dishes coated with collagen and fibronectin, six cell lines (PA, PB, PC, PD, PE and PF) were established from separate pools of papillomas. When tested for tumorigenicity in nude mice by injection of a cell suspension or implantation of cells growing on a plastic liner, two of the lines (PC and PF) produced no tumors at any passage. In contrast, cells of the lines PA and PE produced highly differentiated squamous cell carcinomas from the earliest passage tested. The results with PB and PD were variable on tumorigenicity testing with some passages positive and others negative. When tested for responsiveness to Ca2+ (greater than 0.1 mM) as a differentiation stimulus, all lines responded. In the higher Ca2+ medium there was a 50-95% decrease in colony-forming efficiency, a slight decrease in [3H]thymidine incorporation (except for PA) and an increase in the number of cornified cells (except for early passage PF). Epidermal transglutaminase activity, a marker for terminal differentiation, was increased in the presence of medium with Ca2+ greater than 0.1 mM. However, unlike normal cells, only a fraction of the cells from each of the papilloma-derived cell lines terminally differentiated in response to Ca2+ while the remaining cells continued to proliferate, although at a slower rate. Responsiveness to phorbol ester tumor promoters was also examined in papilloma cell lines. 12-O-Tetradecanoylphorbol-13-acetate (TPA) treatment increased colony forming efficiency, DNA synthesis and colony size in all lines in medium with either 0.05 mM Ca2+ or 1.2 mM Ca2+. TPA treatment also increased ornithine decarboxylase activity in all lines, even at the higher Ca2+ concentration, although normal keratinocytes respond only when grown in medium with low Ca2+. TPA treatment caused only a slight increase in the number of cornified cells and no increase in epidermal transglutaminase activity in papilloma cells while it causes 10-fold or greater increases in these differentiation markers in normal keratinocytes. Thus papilloma cells appear to differ from normal keratinocytes in their ability to maintain a proliferating population under conditions favoring terminal differentiation, their consistent proliferative response to phorbol esters under these same conditions, and their reduced sensitivity to phorbol ester-induced terminal differentiation.(ABSTRACT TRUNCATED AT 400 WORDS)