Mouse Epidermal Carcinogenesis Research Articles

Inhibition of 7,12‐Dimethylbenz[a]anthracene Induced Mouse Skin Carcinogenesis by Artemisia capillaris

Anticarcinogenic activity of medicinal herbs (Artemisia capillaris, Taxus cuspidata, Anthriscus sylveatris, and Curcuma longa) was examined for 7,12-dimethylbenz[a]anthracene (DMBA)-induced mouse skin carcinogenesis. Four types of solvent fractions (hexane, chloroform, ethyl acetate, and butanol) were prepared from the methanolic extract of medicinal herbs. The cytotoxicity and anticarcinogenic activities of solvent fractions were examined for mouse leukemia L1210 cancer cells and for female ICR mouse epidermal carcinogenesis induced by DMBA, respectively. The chloroform fraction of Artemisia capillaris, Taxus cuspidata, and Anthriscus sylveatris was more toxic to L1210 cells than other solvent fractions. The chloroform fraction of Artemisia capillaris markedly reduced the number of tumors/mouse and tumor incidence relative to that of other medicinal herbs tested. Major active chemical constituents in the chloroform fraction of Artemisia capillaries were found to be camphor, 1-borneol, coumarin, and achillin when analyzed by TLC and GC-MS. These results suggest that Artemisia capillaris was the most effective anticarcinogenic medicinal herb for DMBA-induced mouse epidermal carcinogenesis among 4 medicinal herbs tested, and the effect might be attributed to chemical compounds of camphor, 1-borneol, coumarin, and achillin.

P73 expression modulates p63 and Mdm2 protein presence in complex with p53 family-specific DNA target sequence in squamous cell carcinogenesis

The expression of p73 and p63 isoforms is frequently deregulated in human epithelial tumors. We previously showed that loss of p73 protein expression associates with malignant conversion in vivo and ionizing radiation (IR) resistance in vitro in a clonal model of mouse epidermal carcinogenesis. Here we show that loss of endogenous p73 expression in squamous cell carcinoma (SCC) cells and tumors was concomitant with preferential DNA binding of the inhibitory DeltaNp63alpha isoform and reduction of transcriptionally active p63gamma isoforms binding to a p21 promoter sequence in vitro. Reconstitution of TAp73alpha in malignant cells increased the steady state DNA-binding capabilities of the endogenous transcriptionally active TAp63gamma and DeltaNp63gamma isoforms, correlating with restoration of tumor suppression but not IR sensitivity. Loss of p73 in malignant cells also coincided with increased presence of p53 family inhibitor Mdm2 in p53-specific DNA-bound complexes, whereas reconstitution of TAp73alpha expression resulted in exclusion of Mdm2 from these complexes. These results suggest a dual mechanism for TAp73alpha to foster tumor suppression through enhancement of the DNA-binding activity of p63gamma isoforms, and through inhibition of transcriptional repressors Mdm2 or DeltaNp63alpha.

Chromosomal instability and phenotypic plasticity during the squamous–spindle carcinoma transition: association of a specific T(14;15) with malignant progression

In mouse epidermal carcinogenesis, the latest stage of malignant progression involves the transition from squamous cell carcinoma to a highly aggressive type of tumor with spindle morphology. In this work, we have isolated a minor epithelial cell subpopulation (CarC-R) contained in the highly malignant spindle carcinoma cell line CarC. CarC-R exhibited a drastic reduction in tumorigenicity when compared with CarC, but CarC-R-induced tumors were mainly sarcomatoid, although they subsequently reverted to the epithelial phenotype when tumor explants were recultured in vitro. Several single-cell clones with either stable epithelial or fibroblastic phenotypes were isolated from an explanted CarC-R tumor (CarC-RT). All these cell lines contained the same specific point mutation in H-Ras codon 61, but while CarC spindle cells had lost the normal H-Ras allele, it was retained in CarC-R- and CarC-RT-derived cell lines. Furthermore, CarC cells have inactivated p16INK4a and p19INK4a/ARF transcription, while CarC-R and CarC-RT clones expressed p19 mRNA and protein but not p16. Altogether, these results suggest that CarC-R represents a precursor stage to CarC in malignant progression. Spectral karyotyping analysis revealed that CarC-R was highly aneuploid and contained many chromosomal abnormalities. In contrast, CarC had a diploid or tetraploid modal chromosome number and contained a specific T(14;15) translocation in all of the analysed metaphases. The T(14;15) translocation was present in only a minority (1.9%) of CarC-R cells, but it was widely spread in CarC-RT and its derived cell clones, regardless of their epithelial or fibroblastic phenotype, indicating that T(14;15) segregates with malignancy.

RING protein Trim32 associated with skin carcinogenesis has anti-apoptotic and E3-ubiquitin ligase properties.

Tripartite motif protein 32, Trim32, mRNA and protein expression was elevated in independently transformed and tumorigenic keratinocytes of a mouse epidermal carcinogenesis model, in ultraviolet B (UVB)-induced squamous cell carcinomas (SCC), and in approximately 20-25% of chemically induced mouse papillomas and human head and neck SCCs. This suggests that elevated Trim32 expression occurs frequently in experimental epidermal carcinogenesis and is relevant to human cancer. Transduced Trim32 increased colony number in an epidermal in vitro transformation assay and epidermal thickening in vivo when skin-grafted to athymic nu/nu mice. These effects were not associated with proliferation and were not sufficient for tumorigenesis, even with 12-O-tetradecanoylphorbol-13-acetate treatment or defects in the tumor suppressor p53. However, transduced Trim32 inhibited the synergistic effect of tumor necrosis factor alpha (TNFalpha) on UVB-induced apoptosis of keratinocytes in vitro and the apoptotic response of keratinocyte grafts exposed to UVB-light in vivo. Consistent with its RING domain, Trim32 exhibited characteristics of E3-ubiquitin ligases, including being ubiquitylated itself and interacting with ubiquitylated proteins, with increases in these properties following treatment of cultured keratinocytes with TNFalpha/UVB. Interestingly, missense point mutation of human TRIM32 has been reported in Limb-Girdle Muscular Dystrophy Type 2H, an autosomal recessive disease. We propose a model in which Trim32 activities as an E3-ubiquitin ligase favor initiated cell survival in carcinogenesis by blocking UVB-induced TNFalpha apoptotic signaling.

VEGF/VPF overexpression in skin of transgenic mice induces angiogenesis, vascular hyperpermeability and accelerated tumor development

Upregulation of keratinocyte-derived VEGF-A expression has recently been established in non-neoplastic processes of skin such as wound healing, blistering diseases and psoriasis, as well as in skin neoplasia. To further characterize the effects of VEGF-A in skin in vivo, we have developed transgenic mice expressing the mouse VEGF120 under the control of a 2.4 kb 5' fragment of keratin K6 gene regulatory sequences that confers transgene inducibility upon hyperproliferative stimuli. As expected from the inducible nature of the transgene, two of the three founder mice obtained (V27 and V208), showed no apparent phenotype. However, one founder (V2), mosaic for transgene integration, developed scattered red spots throughout the skin at birth. The transgenic offspring derived from this founder developed a striking phenotype characterized by swelling and erythema, resulting in early postnatal lethality. Histological examination of the skin of these transgenics demonstrated highly increased vascularization and edema leading to disruption of skin architecture. Expression of the transgene was silent in adult animals of lines derived from founders V27 and V208. Phorbol ester-induced hyperplasia resulted in transgene induction and increased cutaneous vascularization in adult transgenic mice of these lines. Skin carcinogenesis experiments performed on hemizygous crosses of V208 mice with activated H-ras-carrying transgenic mice (TG.AC) resulted in accelerated papilloma development and increased tumor burden. Previous results from our laboratory showed that VEGF upregulation is a major angiogenic stimulus in mouse epidermal carcinogenesis. By overexpressing VEGF in the skin of transgenic mice we now move a step further toward showing that VEGF-mediated angiogenesis is a rate-limiting step in the genesis of premalignant lesions, such as mouse skin papilloma. Our transgenic mice constitute an interesting model system for in vivo study of the cutaneous angiogenic process and its relevance in tumorigenesis and other skin diseases.

Delays in malignant tumor development in transgenic mice by forced epidermal keratin 10 expression in mouse skin carcinomas.

The keratin cytoskeleton is formed in different epidermal compartments by distinct polypeptides. Basal, proliferative keratinocytes express keratin (K) 5 and K14, whereas, suprabasal, post-mitotic keratinocytes express K1 and K10. Changes in this keratin pattern have been found to occur in hyperproliferative skin disorders and, in particular, throughout mouse epidermal carcinogenesis. Whereas some keratins not found in normal epidermis (K6, K16, K13, and K8) are induced at different stages of tumor development, K1 and K10 expression is lost. To determine whether K1 and K10 loss is just a consequence of the altered differentiation program or an event required for tumor progression, we generated transgenic mice carrying the human keratin 10 gene (hK10) under the control of a bovine keratin 6 gene regulatory region, which is silent in normal skin but is induced and drives transgene expression in hyperproliferative skin keratinocytes and, therefore, in skin tumors. Transgenic animals subjected to a complete carcinogenesis protocol developed tumors that contained various amounts of transgenic hK10. Although no significant difference was found in tumor number or malignancy, tumor onset was significantly delayed in transgenic mice, indicating that the presence of K10 actually impairs tumor development.

EXPRESSION OF INTERMEDIATE FILAMENT PROTEINS BY NORMAL AND TRANSFORMED MOUSE EPIDERMAL-KERATINOCYTES IN CULTURE

The aberrant expression of embryonic simple epithelial keratins K8 and K18 has been previously detected in chemically-induced mouse skin carcinomas and in cultured epidermal keratinocytes containing H-ras oncogenes. In this study, we report that cell lines derived from benign papillomas do not generally synthesize simple epithelial keratins, regardless of the presence of H-ras gene alterations. These results reinforce our previous suggestion that the expression of these keratins during mouse epidermal carcinogenesis is a marker of malignant transformation. The comparison of the two-dimensional electrophoresis intermediate filament protein profiles of transformed keratinocytes in culture, reveal that during in vitro progression to the malignant phenotype increased synthesis of simple epithelial keratins is associated to a general disturbance on the expression of cytoskeletal proteins.

Stage of Tumor Progression, Progressor Agents, and Human Risk

AbstractThat cancer develops from an antecedent process consisting of distinct, definable stages or phases is now considered to be the case in the development of the majority of animal and human neoplasms (1-5). In some of the earliest studies in experimental animals demonstrating the existence of such stages (6-8), two stages, termed initiation and promotion, occurred in that order preceding the development of tumors. The majority of tumors occurrin in the system studied, that of mouse epidermal carcinogenesis, were benign papillomas, and carcinomas developed only later. Subsequently, Foulds (9), from studies on experimental mammary adenocarcinoma, argued that the stage following initiation be termed “progression”, to represent a continuous spectrum of alterations, presumably irreversible, beginning with the initiated cell.

Expression of α6β4 integrin increases during malignant conversion of mouse epidermal keratinocytes: Association of β4 subunit to the cytokeratin fraction

The expression of α6β4 integrin has been analyzed in several keratinocyte cell lines representative of various stages of mouse epidermal carcinogenesis. The immunological analyses carried out show that α6β4 is expressed at the cell surface of the cell lines which exhibit an epithelial or epithelioid morphology. The relative levels of α6β4 expressed at the cell surface increase noticeably from premalignant to malignant cells, as detected by fluorescence flow cytometry. This increase also correlates with the abundance of soluble β4 subunit detected by Western immunoblotting in the different cell lines. However, complete absence of α6β4 has been found in spindle carcinoma cells showing a fibroblast-like phenotype in culture. The integrin remains associated to detergent- and high salt-insoluble cytoskeletal components, organized in stable anchoring contacts, as in human keratinocytes (Carter et al., J. Cell Biol., 111, 3141, 1990). In addition, a significant fraction of the β4 subunit is detected associated to highly purified cytokeratin fractions. These results, together with those regarding the organization of both cellular components in drug-treated cells, support the existence of a close association between α6β4 and the intermediate filaments of the cytoskeleton.

Palm carotene inhibits tumor-promoting activity of bile acids and intestinal carcinogenesis.

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

A role for the E-cadherin cell-cell adhesion molecule during tumor progression of mouse epidermal carcinogenesis.

The expression of the cell-cell adhesion molecules E- and P-cadherin has been analyzed in seven mouse epidermal keratinocyte cell lines representative of different stages of epidermal carcinogenesis. An inverse correlation between the amount of E-cadherin protein and tumorigenicity of the cell lines has been found, together with a complete absence of E-cadherin protein and mRNA expression in three carcinoma cell lines (the epithelioid HaCa4 and the fibroblastoid CarB and CarC cells). A similar result has been detected in tumors induced in nude mice by the cell lines, where induction of E-cadherin expression takes place in moderately differentiated squamous cell carcinomas induced by HaCa4 cells, although at much lower levels than in well-differentiated tumors induced by the epithelial PDV or PDVC57 cell lines. Complete absence of E-cadherin expression has been observed in spindle cell carcinomas induced by CarB or CarC cells. P-cadherin protein was detected in all cell lines that exhibit an epithelial (MCA3D, AT5, PDV, and PDVC57) or epithelioid (HaCa4) morphology, as well as in nude mouse tumors, independent of their tumorigenic capabilities. However, complete absence of P-cadherin was observed in the fibroblast-like cells (CarB and CarC) and in spindle cell carcinomas. The introduction of an exogenous E-cadherin cDNA into HaCa4 cells, or reactivation of the endogenous E-cadherin gene, leads to a partial suppression of the tumorigenicity of this highly malignant cell line. These results suggest a role for E-cadherin in the progression to malignancy of mouse epidermal carcinogenesis. They also suggest that the loss of both E- and P-cadherin could be associated to the final stage of carcinogenesis, the development of spindle cell carcinomas.

An evaluation of the epidermal carcinogenic potential of cutting fluids

Epidermal cancer in humans has been associated with industrial exposure to products derived from unrefined petroleum distillates. Because of this association many of these products, including cutting fluids, are now normally produced from highly refined lubricant base oils. Since highly refined oils are not dermally carcinogenic, one would predict that formulated products prepared from these oils would similarly be non-tumorigenic. However, there are experimental data that suggest that fresh cutting fluids might be dermally carcinogenic and that industrial usage might increase the carcinogenic hazard. The current studies evaluated solvent-extracted lubricant base oils and several products, including fresh and used cutting fluids and certain related industrial oils. These materials were all tested for tumorigenic potential in mouse epidermal carcinogenesis bioassays. None of the solvent-extracted lubricant base oils were dermal carcinogens. Similarly, the cutting fluids and industrial oils formulated from these base oils were not dermally carcinogenic. Finally, there was no evidence that industrial usage influenced the carcinogenic potential of these oils. It was concluded that solvent-extracted oils and the products derived from these oils pose minimal skin cancer hazards when used under appropriate conditions in industrial settings.

A 90-Day Toxicity Study of the Effects of Petroleum Middle Distillates on the Skin of C3H Mice

Petroleum middle distillates (PMDs) elicit skin tumors in mouse epidermal carcinogenesis studies. The response is characterized by a long latency with only a small percentage of animals developing tumors. Although the carcinogenic activity of certain other petroleum hydrocarbons largely depends upon the presence of polycyclic aromatic hydrocarbons (PAHs), many PMDs contain relatively low concentrations of PAHs. PMDs are also irritating to mouse skin, and chronic irritation may be involved in the development of skin tumors. This study was conducted to investigate the patterns of cutaneous irritation elicited by topical application of PMDs having compositional differences. The three PMDs selected for study were a steam cracked gas oil (SCGO), a lightly refined paraffinic oil (LRPO), and a jet fuel (JF). Male C3H/HeNCr1BR mice (25/group) were treated topically (37.5 microliters 2x/week for 13 weeks) with 10%, 50% or 100% (undiluted) concentrations of each PMD. Catalytically cracked clarified oil (CCCO, 10%), a potent carcinogen to mouse skin, was also tested. The vehicle was a noncarcinogenic mineral oil with a viscosity of 90 SUS. Cutaneous changes were evaluated by gross observations and light microscopy. Cutaneous irritation was the only significant toxic response in this study. Neither the vehicle nor any of the 10% PMD concentrations produced significant cutaneous irritation. The 10% CCCO and 50% PMD treatments all elicited slight to moderate proliferative and inflammatory changes in mouse skin. Ulcers were also observed microscopically in mice treated with 10% CCCO and 50% SCGO. The 100% SCGO treatment produced evidence of necrosis on Days 1-7 but not later in the study despite continued treatment. In contrast, the irritating effects of 100% LRPO were not evident until 2-3 weeks of study, and at study completion were characterized by moderately severe inflammatory and proliferative changes. The effects of 100% JF were qualitatively similar to 100% LRPO but less marked. Thus, the SCGO caused a different pattern of cutaneous responses than either LRPO or JF. The possible relationships of these cutaneous changes to epidermal carcinogenesis are being studied further.

Anticarcinogens from fried ground beef: heat-altered derivatives of linoleic acid.

Fried ground beef contains substances that inhibit mutagenesis in bacteria and the initiation of epidermal carcinogenesis in mice by 7,12-dimethylbenz [a]anthracene (DMBA). The inhibitors apparently act at least in part via inhibition of cytochrome P-450 activity. A highly purified fraction that inhibited cytochrome P-450 activity in vitro was isolated by HPLC and characterized by GC-MS, and by UV and proton NMR spectroscopy. The fraction contained four isomeric derivatives of linoleic acid each containing a conjugated double-bond system (designated CLA). Synthetically prepared CLA (containing all four isomers) was tested for anti-initiation activity in the two-stage mouse epidermal carcinogenesis system. Seven days, 3 days and 5 min prior to DMBA application, CLA was applied at doses of 20, 20 and 10 mg respectively. Control mice were treated similarly with linoleic acid or solvent (acetone). One week after initiation, and twice weekly thereafter, all mice were treated with 12-O-tetradecanoylphorbol-13-acetate to effect tumor promotion. There was no difference in tumor incidence or yield between linoleic acid-treated mice and solvent-treated control mice. By contrast, the CLA-treated mice developed only about half as many papillomas and exhibited a lower tumor incidence compared with the control mice.

Evaluation of the dermal carcinogenic potential of Tar sands bitumen-derived liquids

The carcinogenic potential of Athabasca tar sands and six experimental liquids derived from crude bitumen was evaluated utilizing the mouse epidermal carcinogenesis model. Tar sands, bitumen, and untreated naphtha produced few, if any, tumors. Three thermally and catalytically cracked liquids, light (nominal boiling range: 149–316°C) and heavy (nominal boiling range: >316°C) gas oils and gas oil blend (boiling range: >316°C), produced a significant number of epidermal neoplasms. A synthetic crude oil, prepared by blending naphtha and light and heavy gas oils, was moderately carcinogenic; however, the activity of this sample fell within the range of values obtained in studies of crude petroleum samples. Since the bitumen-derived streams do not differ substantially in carcinogenic potency from petroleum-derived materials of comparable boiling range and process history, industrial hygiene practices which limit exposures to levels comparable to those observed in the petroleum-refining industry should provide similar measures of protection.

Evaluation of the Dermal Carcinogenic Potential of Tar Sands Bitumen-Derived Liquids

Evaluation of the Dermal Carcinogenic Potential of Tar Sands Bitumen-Derived Liquids. MCK.EE, R. H., STUBBLEFIELD, W. A., LEWIS, S. C, SCALA, R. A., SIMON, G. S., AND DEPASS, L. R. (1986). Fundam Appl. Toxwoi 7, 228−235. The carcinogenic potential of Athabasca tar sands and six experimental liquids derived from crude bitumen was evaluated utilizing the mouse epidermal carcinogenesis model. Tar sands, bitumen, and untreated naphtha produced few, if any, tumors. Three thermally and catalytically cracked liquids, light (nominal boiling range: 149-316°C) and heavy (nominal boiling range: >316°C) gas oils and gas oil blend (boiling range' >316°C), produced a significant number of epidermal neoplasms. A synthetic crude oil, prepared by blending naphtha and light and heavy gas oils, was moderately carcinogenic; however, the activity of this sample fell within the range of values obtained in studies of crude petroleum samples. Since the bitumen-derived streams do not differ substantially in carcinogenic potency from petroleum-denved materials of comparable boiling range and process history, industrial hygiene practices which limit exposures to levels comparable to those observed in the petroleum-refining industry should provide similar measures of protection.