The Tg.AC (v-Ha-ras) transgenic mouse: nature of the model.

The Tg.AC (v-Ha-ras) transgenic mouse model provides a reporter phenotype of skin papillomas in response to either genotoxic or nongenotoxic carcinogens. In common with the conventional bioassay, the Tg.AC model responds to known human carcinogens and does not respond to noncarcinogens. It also does not respond to most chemicals that are positive in conventional bioassays principally at sites of high spontaneous tumor incidence. The mechanism of response of the Tg.AC model is related to the structure and genomic position of the transgene and the induction of transgene expression through specific mediated interactions between the chemicals and target cells in the skin.

Tg.AC genetically altered mouse: assay working group overview of available data.

In a Government/Industry/Academic partnership to evaluate alternative approaches to carcinogenicity testing, 21 pharmaceutical agents representing a variety of chemical and pharmacological classes and possessing known human and or rodent carcinogenic potential were selected for study in several rodent models. The studies from this partnership project, coordinated by the International Life Sciences Institute, provide additional data to better understand the models' limitations and sensitivity in identifying carcinogens. The results of these alternative model studies were reviewed by members of Assay Working Groups (AWG) composed of scientists from government and industry with expertise in toxicology, genetics, statistics, and pathology. The Tg.AC genetically manipulated mouse was one of the models selected for this project based on previous studies indicating that Tg.AC mice seem to respond to topical application of either mutagenic or nonmutagenic carcinogens with papilloma formation at the site of application. This communication describes the results and AWG interpretations of studies conducted on 14 chemicals administered by the topical and oral (gavage and/or diet) routes to Tg.AC genetically manipulated mice. Cyclosporin A, an immunosuppresant human carcinogen, ethinyl estradiol and diethylstilbestrol (human hormone carcinogens) and clofibrate, an hepatocarcinogenic peroxisome proliferator in rodents, were considered clearly positive in the topical studies. In the oral studies, ethinyl estradiol and diethylstilbestrol were negative, cyclosporin was considered equivocal, and results were not available for the clofibrate study. Of the 3 genotoxic human carcinogens (phenacetin, melphalan, and cyclophosphamide), phenacetin was negative by both the topical and oral routes. Melphalan and cyclophosphamide are, respectively, direct and indirect DNA alkylating agents and topical administration of both caused equivocal responses. With the exception of clofibrate, Tg.AC mice did not exhibit tumor responses to the rodent carcinogens that were putative human noncarcinogens, (di(2-ethylhexyl) phthalate, methapyraline HCl, phenobarbital Na, reserpine, sulfamethoxazole or WY-14643, or the nongenotoxic, noncarcinogen, sulfisoxazole) regardless of route of administration. Based on the observed responses in these studies, it was concluded by the AWG that the Tg.AC model was not overly sensitive and possesses utility as an adjunct to the battery of toxicity studies used to establish human carcinogenic risk.

Age-dependent skin tumorigenesis and transgene expression in the Tg.AC (v-Ha-ras) transgenic mouse.

Transgenic Tg.AC (v-Ha-ras ) mice develop skin tumors in response to specific carcinogens and tumor promoters. The Tg.AC mouse carries the coding sequence of v-Ha ras, linked to a zeta-globin promoter and an SV40 polyadenylation signal sequence. The transgene confers on these mice the property of genetically initiated skin. This study examines the age-dependent sensitivity of the incidence of skin papillomas in Tg.AC mice exposed to topically applied 12-O:-tetradecanoylphorbol-13-acetate (TPA) treatment, full thickness skin wounding or UV radiation. Skin tumor incidence and multiplicity were strongly age-dependent, increasing with increasing age of the animal when first treated at 5, 10, 21 or 32 weeks of age. Furthermore, the temporal induction of transgene expression in keratinocytes isolated from TPA-treated mouse skin was also influenced by the age of the mice. Transgene expression was seen as early as 14 days after the start of TPA treatment in mice that were 10-32 weeks of age, but was not detected in similarly treated 5-week old mice. When isolated keratinocytes were fractionated by density gradient centrifugation the highest transgene expression was found in the denser basal keratinocytes. Transgene expression could be detected in the denser keratinocyte fraction as early as 9 days from start of TPA treatment in 32-week old mice. Using flow cytometry, a positive correlation was observed between expression of the v-Ha-ras transgene and enriched expression of the cell surface protein beta1-integrin, a putative marker of epidermal stem cells. This result suggests that, in the Tg.AC mouse, an age-dependent sensitivity to tumor promotion and the correlated induction of transgene expression are related to changes in cellular development in the follicular compartment of the skin.

Responses of transgenic mouse lines p53(+/-) and Tg.AC to agents tested in conventional carcinogenicity bioassays.

The haplo-insufficient p53 knockout (p53+/-) and zetaglobin v-Ha-ras (Tg.AC) transgenic mouse models were compared to the conventional two rodent species carcinogen bioassay by prospectively testing nine chemicals. Seven of the chemicals classified as carcinogens in the conventional bioassay induced tumors in the liver or kidneys of B6C3F1 mice, and one (pentachlorophenol) also induced tumors in other tissues. Only three chemicals, furfuryl alcohol, pyridine, and pentachlorophenol, induced tumors in rats. The tumorigenic effect of pyridine was seen in F344 rats but not in Wistar strain rats. None of the chemicals induced tumors in the p53+/- transgenic mice, which is consistent with the absence of genotoxicity of these chemicals. Only two of the seven nongenotoxic carcinogens were positive in the Tg.AC model (lauric acid diethanolamine and pentachlorophenol). These results show that these transgenic models do not respond to many chemicals that show strain- or species-specific responses in conventional bioassays.

Oral administration of dimethylvinyl chloride increases frequency of forestomach papillomas in Tg.AC mice.

This work was initiated to determine the potential for the Tg.AC mouse model to identify chemical carcinogens by an oral route of administration. Tg.AC v-Ha-ras transgenic mice were exposed to dimethyvinyl chloride (DMVC; 1-chloro-2-methylpropene), a structural analog of the human carcinogen vinyl chloride. In the National Toxicology Program 2-yr bioassay, DMVC induced tumors in the oral, nasal, and gastric epithelia of rats and mice. Initial studies were performed in female Tg.AC mice to determine an appropriate oral dose of DMVC to evaluate the potential for stratified gastric or oral epithelia of Tg.AC mice to serve as a target tissue for a transgene-dependent induced tumorigenic response. DMVC was administered to 13- to14-wk-old Tg.AC mice by gavage at doses of 0, 50, 100, and 200 mg/kg five times a week for 20 wk. The forestomachs of DMVC-treated Tg.AC mice had an increasing number of papillomas, which were associated with an increase in the dose of DMVC. The average numbers of papillomas per mouse per dose were 2.4, 7.6, 14.1, and 12.6 for the 0, 50, 100, and 200-mg/kg dose groups, respectively. The optimum papillomagenic dose of 100 mg/kg DMVC was established and administered for 5, 10, and 15/wk to investigate the kinetics of papilloma induction in Tg.AC mice. The average numbers of papillomas per animal were 1.8, 8.8, and 19.0 at 5, 10, and 15 wk, respectively. Reverse transcription-polymerase chain reaction assays determined that the v-Ha-ras transgene was transcriptionally active in all tumor tissues but not in nontumor tissues. In situ hybridization assays performed in conjunction with bromodeoxyuridine in vivo labeling localized the transgene-expressing cells of the forestomach papillomas to the proliferating cellular component of the tumors, as previously seen in skin papillomas of Tg.AC mice. The present results confirm that DMVC is tumorigenic and that oral routes of administration can be used to rapidly elicit a transgene-associated tumor response in the forestomach of Tg.AC mice.

Development of a transgenic mouse model for carcinogenesis bioassays: evaluation of chemically induced skin tumors in Tg.AC mice.

Transgenic rodent models have emerged as potentially useful tools in the assessment of drug and chemical safety. The transgenic Tg.AC mouse carries an inducible v-Ha-ras oncogene that imparts the characteristic of genetically initiated skin to these animals. The induction of epidermal papillomas in the area of topically applied chemical agents, for duration of not more than 26 weeks, acts as a reporter phenotype that defines the activity of the test article. We describe here the activity of six chemicals that have been previously characterized for activity in the standard 2-year bioassay conducted by the National Toxicology Program (NTP). Homozygous female Tg.AC mice were treated with benzene (BZ), benzethonium chloride (BZTC), o-benzyl-p-chlorophenol (BCP), 2-chloroethanol (2-CE), lauric acid diethanolamine (LADA) and triethanolamine (TEA). BZ and LADA induced skin papillomas in a dose-dependent manner, while BCP induced papillomas only at the highest dose. BZTC, 2-CE, and TEA exhibited no activity. The correspondence of chemical activity in Tg.AC mice with that in the 2-year bioassay was high. A comparison of responsiveness to BZ and LADA was made between hemizygous and homozygous female Tg.AC mice. Both genotypes appear to be equally sensitive to maximum doses of active compounds. The results reported here indicate that the Tg.AC transgenic mouse model can discriminate between carcinogens and noncarcinogens and that both mutagenic and nonmutagenic chemicals can be detected. These studies provide support for the adjunctive use of the Tg.AC transgenic mouse skin tumor model in drug and chemical safety assessment and for the prediction of the carcinogenic potential of chemicals.

Morphological characterization of spindle cell tumors induced in transgenic Tg.AC mouse skin.

Transgenic Tg.AC mice carry a v-Ha-ras coding region flanked by a zeta-globin promoter and an SV40 polyadenylation signal sequence. These mice respond to carcinogens by developing epidermal papillomas. In some cases, malignancies develop at the sites of these papillomas. Various patterns of squamous cell differentiation were observed in these malignancies. One malignancy that developed at the site of the papillomas was composed of bundles of spindle cells. This lesion is difficult to distinguish from fibrosarcomas by light microscopy. We characterized 16 of these malignancies (tentatively classified as spindle cell tumors) to determine if they were of epithelial or mesenchymal origin. Papillomas were induced in Tg.AC mice by full thickness wounding, 12-O-tetradecanoyl-13-phorbol acetate treatment, or ultraviolet radiation. With time, some papillomas became broad-based, downwardly invading lesions. These lesions were examined by light microscopy with immunohistochemical analysis for cytokeratins and by electron microscopy. Immunohistochemical examination with a polyclonal anti-cytokeratin antibody demonstrated various degrees of keratin staining in all tumors examined. Attenuated desmosomes were also observed in these lesions by electron microscopy. These results indicate an epithelial origin for these malignancies; therefore, they should be classified as spindle cell carcinomas.

TGF alpha is dispensable for skin tumorigenesis in Tg.AC mice.

Alterations in growth factor signaling pathways frequently accompany the development and maintenance of epithelial neoplasia. Transforming growth factor alpha (TGF alpha) and its epidermal growth factor receptor have been thought to play an especially important role in epithelial neoplasia. In this study, mice were derived genetically deficient (null) in functional TGF alpha expression and carrying the Tg.AC/v-Ha-ras transgene. The goals were to determine if (a) papillomagenesis was dependent on TGF alpha and (b) progression to malignancy was dependent on TGF alpha expression. Groups of male and female mice heterozygous or homozygous for the TGF alpha null allele and hemizygous for the Tg.AC transgene were treated twice weekly for 10 or 15 wk with doses of 12-O-tetradecanoylphorbol-13-acetate (TPA) known to produce papillomas in Tg.AC mice. Papillomas were readily induced in both male and female TGF alpha null mice. Malignant progression of papillomas was observed in all TGF alpha null treatment groups. Additionally, we examined the response of TGF alpha null mice to full thickness dorsal wounds, a stimulus known to promote papillomagenesis in Tg.AC mice. As in the TPA study, papillomas were induced in both male and female TGF alpha null mice. These studies indicate that TGF alpha is not required for the induction and maintenance of papillomas nor is it essential for the malignant conversion of papillomas in Tg.AC mice.

Comparative expression of novel vascular endothelial growth factor/vascular permeability factor transcripts in skin, papillomas, and carcinomas of v-Ha-ras Tg.AC transgenic mice and FVB/N mice.

One of the most frequently detected changes in human solid tumors is the mutation of the ras oncogene, which has been associated with production of angiogenic growth factors such as vascular endothelial growth factor/vascular permeability factor (VEGF/VPF). Using the v-Ha-ras Tg-AC transgenic mice and the background FVB/N strain of inbred mice, the pattern of expression of specific VEGF/VPF transcripts was characterized in major organs and in skin, papillomas, and carcinomas during multi-stage skin carcinogenesis. Three VEGF/VPF transcripts were found to be constitutively expressed in skin as well as the major organs in both mouse strains, which corresponded in size and sequence to previously reported murine VEGF120 with a bp size of 331, VEGF164 with a bp size of 333, and VEGF188 with a bp size of 407. A previously unreported fourth murine transcript was also detected in skin and major tissues from both mouse strains which corresponded to rat VEGF144, with a bp size of 404. In addition, a unique 425 bp VEGF transcript which corresponded to human VEGF205 was present in highly vascularized tissues including heart, lung, liver, kidney, brain, as well in papillomas and carcinomas isolated from v-Ha-ras Tg.AC mice. In contrast, VEGF205 was present only in carcinomas derived from FVB/N mice. An antibody generated from a peptide sequence designed to detect each of the five VEGF/VPF peptides defined by RT-PCR analysis confirmed the existence of these five peptides and confirmed that the murine VEGF205 peptide was selectively expressed in papillomas and carcinomas derived from v-Ha-ras Tg.AC mice. These results demonstrate that there is significant alternative splicing of the murine VEGF/VPF gene during multi-stage carcinogenesis, which results in four commonly expressed VEGF transcripts. In addition, these studies identified a fifth VEGF transcript and peptide at the later stages of tumor promotion and in progression which appears to be linked to the presence of v-Ha-ras.

Induction of transgene expression in Tg.AC(v-Ha-ras) transgenic mice concomitant with DNA hypomethylation.

Tg.AC transgenic mice have a transgene composed of a zeta-globin transcriptional control region, a v-Ha-ras coding region, and a simian virus 40 3' polyadenylation signal sequence. Induced ectopic expression of the transgene by chemical treatment or full-skin-thickness wounding leads to the development of skin papillomas. Reverse transcription-polymerase chain reaction assays and protein blotting indicated that the transgene was expressed 16-28 d after full-skin-thickness surgical wounding. Normal unwounded skin did not express the transgene. DNA blotting indicated that the position of the transgene remained stable during wound-induced tumorigenesis. Concomitant with the v-Ha-ras mRNA and protein expression was the hypomethylation of specific MspI/HpaII sites within the transgene. These results are consistent with the hypothesis that hypomethylation is required for the induced and sustained expression of the Tg.AC v-Ha-ras transgene in spontaneous and induced tumors in Tg.AC mice.

Hemizygous Tg.AC transgenic mouse as a potential alternative to the two-year mouse carcinogenicity bioassay: evaluation of husbandry and housing factors.

The dermal Tg.AC transgenic mouse model has been proposed as a potential alternative to the conventional (e.g. oral, dermal, parenteral, inhalation, etc.) 2-year rodent bioassay for detecting chemical carcinogenicity. The present study was designed to address a number of technical aspects of this model as well as to augment the database being developed with the Tg.AC system at the NIEHS. Hemizygous Tg.AC mice were implanted s.c. with microchips for identification and housed individually in polycarbonate (i.e. 'plastic') or suspended stainless-steel wire-bottom (i.e. 'metal') cages. Treatment consisted of dermal application of the test or control material in treatment volumes of 200 microl of acetone. Groups of 10 males and 10 females were treated as follows: G1--shaved, no treatment; G2--acetone control seven times a week; G3--100 microl of benzene three times a week; G4--150 microl of benzene three times a week; G5--1.25 microg of phorbol ester (PMA) twice a week. The G1-G5 mice were housed in plastic caging with Alpha-dri bedding. Three additional groups were housed in stainless-steel wire-bottom caging: G6--shaved, no treatment; G7--acetone control seven times a week; G8--1.25 microg of PMA twice a week. The PMA-treated mice (G5 and G8) served as the positive controls. Mice were treated for 20 weeks followed by a 6-week recovery period prior to necropsy. The incidence of dermal papillomas in the shaved area was recorded weekly. There were no spontaneous papillomas in the target area of any of the untreated (G1) or vehicle control (G2) animals in the polycarbonate cages. One papilloma was observed in the untreated mice (G6) and one in the vehicle control group (G7) in the steel cages. This suggests that the type of caging, the shaving process, microchip implantation and daily acetone treatment for 20 weeks are all consistent with a very low background incidence of papillomas in this model. Papillomas were observed in the positive control groups as early as 4 weeks of treatment and increased both in number per mouse and number of mice affected up to a maximum average of 3.5 papillomas per mouse and 55% (11/20) mice with papillomas in G5 and 2.7 and 80% (16/20) in G8. A plateau was reached at about week 13 and the numbers of papillomas remained stable through the rest of the treatment and recovery phases. The low dose of benzene (100 microl) showed no significant effect, whereas the higher dose (150 microl) produced a moderate number of papillomas beginning at about week 11. The results of this study are comparable with earlier studies at the NIEHS and indicate reproducibility between laboratories and that the Tg.AC transgenic mouse model is suitable for use in an industrial pre-clinical safety evaluation context.

The transgenic Tg.AC mouse model for identification of chemical carcinogens.

The Tg.AC (zetaglobin promoted v-Ha-ras) transgenic mouse is being evaluated as a short-term carcinogenicity bioassay. In order to harmonize the evaluation effort in diverse laboratories, an operational bioassay protocol has been established. Data, based principally on retrospective assay of known carcinogens or tumor promoters and non-carcinogens, are presented that support the operational protocol. The Laboratory of Environmental Carcinogenesis and Mutagenesis at the NIEHS has been evaluating transgenic rodent models for utility in differentiating carcinogens from non-carcinogens. Our main approach in this method development effort has been to retrospectively study responses of the models to chemicals of known rodent carcinogenic potential. To this end we have tested mainly chemicals that have been previously studied in chronic rat and/or mouse bioassays by the National Toxicology Program. Development of the data base and assessment of the utility of the models will be immeasurably aided by the availability of a standardized experimental protocol. The purpose of this communication is to present the elements of the Laboratory of Environmental Carcinogenesis and Mutagenesis Tg.AC mouse bioassay protocol and to show experimental results that led to the development of our study design.

Kinetics of wound-induced v-Ha-ras transgene expression and papilloma development in transgenic Tg.AC mice.

The Tg.AC transgenic mouse, which harbors an activated v-Ha-ras coding region that is fused to an embryonic zeta globin transcriptional control region and a 3' simian virus 40 polyadenylation sequence, rapidly develops epidermal papillomas in response to topical application of chemical carcinogens or tumor promoters or to full-thickness wounding of the dorsal skin. In this report, we investigated the localization and temporal induction of v-Ha-ras transgene expression after full-thickness wounding of Tg.AC mouse skin. Surgically inflicted full-thickness incisions 3 cm long yielded four to six papillomas per Tg.AC mouse by 5 wk after wounding. Similar wounding of the FVB/N isogenic host strain did not produce tumors, which implicates a causal role for the v-Ha-ras transgene. Reverse transcription-polymerase chain reaction assays detected the v-Ha-ras transgene transcript in total RNA samples isolated from wound-associated tissue 3 and 4 wk after wounding. Tissues 1-2 wk after wounding and all non-wound-associated tissues were negative for transgene expression. In situ hybridization experiments using transgene-specific 35S-labeled antisense RNA probes localized transgene expression to the basal epidermal cells in wound-induced papillomas. Adjacent normal and hyperplastic skin tissues were negative for transgene expression by this assay. This work supports the hypothesis that the wound repair response leads to the transcriptional activation and continued expression of the v-Ha-ras transgene in specific cells in the skin, which alters normal epithelial differentiation and ultimately results in neoplastic growth.

Identifying chemical carcinogens and assessing potential risk in short-term bioassays using transgenic mouse models.

Cancer is a worldwide public health concern. Identifying carcinogens and limiting their exposure is one approach to the problem of reducing risk. Currently, epidemiology and rodent bioassays are the means by which putative human carcinogens are identified. Both methods have intrinsic limitations: they are slow and expensive processes with many uncertainties. The development of methods to modify specific genes in the mammalian genome has provided promising new tools for identifying carcinogens and characterizing risk. Transgenic mice may provide advantages in shortening the time required for bioassays and improving the accuracy of carcinogen identification; transgenic mice might now be included in the testing armamentarium without abandoning the two-year bioassay, the current standard. We show that mutagenic carcinogens can be identified with increased sensitivity and specificity using hemizygous p53 mice in which one allele of the p53 gene has been inactivated. Furthermore, the TG.AC transgenic model, carrying a v-Ha-ras construct, has developed papillomas and malignant tumors in response to a number of mutagenic and nonmutagenic carcinogens and tumor promoters, but not to noncarcinogens. We present a decision-tree approach that permits, at modest extra cost, the testing of more chemicals with improved ability to extrapolate from rodents to humans.

Odontogenic tumours in the v-Ha-ras (TG.AC) transgenic mouse.

A line of homozygous transgenic mice (TG.AC) carrying a v-Ha-ras gene fused to the promoter of the zeta globin gene produces a variety of mesenchymal and epithelial neoplasms including odontogenic tumours. The 1-year incidence of odontogenic tumour formation in these mice was approx. 35%. Tumours formed more often in the mandible than maxilla. The various types of tumours frequently presented with: (1) primarily mesenchymal cells in a dense fibrous-like matrix, or (2) loose stroma surrounded by anastomosing cords of epithelial cells that exhibited squamous differentiation, or (3) odontomas forming mineralized tooth structures by well-differentiated odontoblasts and ameloblasts. Some tumours had areas with all three of these characteristics. Mineralized dentine and enamel in the odontomas were morphologically similar to those of normal murine teeth. Odontogenic tumours expressed the v-Ha-ras transgene that was primarily localized to the mesenchymal cells. Proliferating-cell nuclear antigen immunohistochemistry showed that the mesenchymal cells adjacent to the epithelial cords not only expressed the ras transgene but were also actively proliferating. The TG.AC mouse provides an excellent model for the study of odontogenic tumours and tooth development.

Genetic alterations cooperate with v-Ha-ras to accelerate multistage carcinogenesis in TG.AC transgenic mouse skin.

TG.AC transgenic mice harbor a v-Ha-ras transgene and retain two normal c-Ha-ras alleles and are susceptible to skin tumor formation by 12-O-tetradecanoylphorbol-13-acetate (TPA). To determine whether normal c-Ha-ras antagonizes the oncogenic potential of the v-Ha-ras transgene and/or whether additional non-Ha-ras 7,12-dimethylbenz(a)anthracene (DMBA) initiation target genes exist in mouse skin, which could cooperate with v-Ha-ras to increase the frequency of initiation, rate of promotion, or risk of malignant conversion, we treated TG.AC mouse skin with a single subthreshold dose of DMBA. This was followed by limited TPA or diacylglycerol promotion to select for cells with additional genetic alterations over those cells containing the v-Ha-ras transgene only. DMBA-treated/TPA-promoted TG.AC mice demonstrated a 10-fold increase in the average number of papillomas per mouse, a greater incidence of papilloma bearing-mice, and an increased papilloma growth rate when compared to acetone-treated/TPA-promoted TG.AC mice. These profound changes in papilloma frequency and growth occurred in the absence of the characteristic DMBA-induced A182-->T mutation in c-Ha-ras and immunohistochemical nuclear staining for p53 protein. DMBA-treated/acetone-promoted TG.AC mice did not develop any tumors. Limited promotion with the model diacylglycerol, sn-1,2-didecanoylglycerol, similarly produced an average of 10-fold more papillomas in DMBA-treated mice than in acetone-treated/sn-1,2-didecanoylglycerol-promoted TG.AC mice. DMBA-treated/TPA-promoted TG.AC mice developed their first malignancy by 16 weeks, and by 30 weeks, 50% of the mice developed malignancies, whereas no malignancies were observed in acetone-treated/TPA-promoted TG.AC mice. These results indicate that there exist unidentified DMBA initiation target genes in TG.AC mouse skin that cooperate with mutant Ha-ras to increase papilloma frequency, growth, and malignant conversion, and that promoter treatment can influence malignant conversion by selecting for cells with multiple genetic alterations.

A transgenic mouse model (TG.AC) for skin carcinogenesis: inducible transgene expression as a second critical event.

The v-Ha-ras transgenic TG.AC mouse line behaves as a genetically initiated model for mouse skin tumorigenesis with enhanced susceptibility to skin carcinogens. TG.AC mice develop epidermal papillomas in fewer than 20 weeks in response to the topical application of a variety of chemicals such as complete carcinogens, phorbol ester-type tumor promoters, and nonphorbol ester-type tumor promoters as well as to full-thickness skin wounds or plucking of the dorsal hair. We have found that the pedunculated epidermal papillomas can arise as focal hyperplasias from the permanent portion of the follicular epithelium. Expression of the v-Ha-ras transgene serves as a marker for tumor development since it is expressed at significant levels in the papilloma precursors, focal follicular hyperplasias, and the papillomas but not in the surrounding skin. Transgene expression colocalizes with increased cell proliferation in the papillomas as compared to non-tumor bearing surrounding skin. Malignant skin tumors, primarily squamous cell carcinomas and sarcomas, develop from sites of papilloma development in approximately 40% of papilloma bearing mice. As well as significant levels of transgenic v-Ha-ras expression, some of the malignancies also exhibit karyotypic changes which include trisomy of chromosome six or fifteen, but not chromosome seven. We believe that the TG.AC mouse line serves not only as a model for studying the mechanisms of skin tumorigenesis, but will also be a useful adjunct to the two year NTP toxicity/carcinogenicity studies by identifying carcinogens in fewer than 20 weeks.

Transformation of BALB/c-3T3 cells: IV. Rank-ordered potency of 24 chemical responses detected in a sensitive new assay procedure.

This report introduces an improved method of detecting chemical-induced morphological transformation of A-31-1-13 BALB/c-3T3 cells. The new procedure uses an increased target cell population to assess chemical-induced damage by increasing the initial seeding density and by delaying the initiation time of chemical treatment. Furthermore, a newly developed co-culture clonal survival assay was used to select chemical doses for the transformation assay. This assay measured the relative cloning efficiency (RCE) of chemical treatments in high-density cell cultures. In addition, transformation assay sensitivity was enhanced through the use of improved methods to solubilize many chemicals. From a group of 24 chemicals tested in at least two trials, clear evidence of chemical-induced transformation was detected for 12 chemicals (aphidicolin, barium chloride-2H2O, 5-bromo-2'-deoxyuridine, C.I. direct blue 15, trans-cinnamaldehyde, cytosine arabinoside, diphenylnitrosamine, manganese sulfate-H2O, 2-mercaptobenzimidazole, mezerein, riddelliine, and 2,6-xylidine); 2 chemicals had equivocal activity [C.I. direct blue 218 and mono(2-ethylhexyl)phthalate], 9 chemicals were inactive [carisoprodol, chloramphenicol sodium succinate, 4-chloro-2-nitroaniline, C.I. acid red 114, isobutyraldehyde, mono(2-ethylhexyl)adipate, sodium fluoride, and 12-O-tetradecanoylphorbol-13-acetate), and 1 chemical had an indeterminate response (2,6-dinitrotoluene). All positive responses were detected in the absence of an exogenous activation system and exhibited significant activity at two or more consecutive doses. This report also presents a mathematical method that uses t-statistics for rank-ordering the potency of chemical-induced transformation responses. This model detects sensitivity differences in experiments used to evaluate chemical-induced transformation. Furthermore, it provides a method to estimate a chemical's transformation response in terms of the historical behavior of the assay, as well as its future activity. The most active of the 24 chemicals was mezerein, and the least active chemical was diphenylnitrosamine.