Gap-junctional intercellular communication in epidermal cell lines from selected stages of SENCAR mouse skin carcinogenesis

Homologous and heterologous gap-junctional intercellular communication (IC) was characterized in a panel of cell lines derived from selected stages of SENCAR mouse skin carcinogenesis. This panel included a "carcinogen-altered" cell line, 3PC, obtained from Ca2+-resistant primary adult keratinocytes after exposure to dimethylbenz(a)anthracene as well as cell lines obtained from early and late-stage papillomas and a squamous cell carcinoma (CA3/7) generated during standard in vivo initiation/promotion protocols (dimethylbenz(a)anthracene/12-O-tetradecanoyl-phorbol-13-acetate). Also studied was a cell line (B66BA) obtained from a metastatic lesion following benzo(a)pyrene-induced skin tumorigenesis. Intercellular communication was measured in low-calcium (0.05 mM) medium by quantitation of cell-cell transfer of microinjected fluorescent dye Lucifer Yellow CH. Homologous IC ability diminished progressively from 68 dye-coupled cells per injection for 3PC cultures, to between 21 and 54 dye-coupled cells per injection for three papilloma-derived cell lines, to six and three dye-coupled cells per injection for CA3/7 and B66BA cells, respectively. To test communication of these cells with their normal counterparts, heterologous IC was examined in cocultures with primary adult keratinocytes. Under the conditions used, normal cells established functional communication channels with each cell line tested, showing no selectivity. These results suggest that progressive loss of homologous but not heterologous IC capacity accompanies neoplastic development in mouse skin carcinogenesis.     

Metastatic potential of mouse skin carcinomas produced by different protocols of chemical carcinogenesis.

Squamous cell carcinomas (SCC) of the mouse skin were produced by three different protocols of chemical carcinogenesis, i.e., complete carcinogenesis with 7,12-dimethylbenz(a)anthracene (DMBA) two-stage carcinogenesis with DMBA as initiator, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) as promoter and three stage carcinogenesis with DMBA, TPA and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) as third-stage agent or progressor. Tumors were sequentially studied at weeks 38-52 of treatment. Although no significant differences in the rate of appearance of gamma-glutamyl transpeptidase (GGT) could be seen, a larger number of SCC produced by complete carcinogenesis protocols were GGT-negative. This coincided with the higher grade of malignancy of these tumors as evaluated by histopathology. In general terms high-grade tumors were seen more frequently in the complete carcinogenesis experiment than in the other two protocols. SCC produced by complete carcinogenesis also exhibited a markedly higher DNA index than the SCC from the other experimental groups. All three protocols were very effective in producing late metastasizing tumors, and no significant differences could be established in the incidence of spontaneous lung metastasis. This shows that, contrary to general knowledge, if adequately observed for more than 40 weeks, SCC of the murine skin is able to metastasize in the lung in approximately 30% of cases. Nevertheless, complete carcinogenesis-induced SCC were usually of higher histological grade, a proportion of these were GGT-negative and produced more multiple or diffuse metastases than the tumors induced by the multistage protocols.     

Multistage chemical carcinogenesis protocols produce spindle cell carcinomas of the mouse skin

Spindle cell carcinomas were identified using polyacrylamidegel electrophoresLs and immunoblotting of proteins extractedfrom paraffin-embedded tissue sections. Immunohisto-chemistryusing rabbit monospecific antisera against the mouse 55 kd keratinpolypeptide also identified these tumors. A group of 53 SENCARmice initiated with 7,12-dimethyl-benz[a]anthracene (DMBA) andpromoted with 12-O-tetra-decanoylphorbol-13-acetate (TPA) yielded,after one year, four spindle cell carcinomas (0.07/mouse), whereasanother group of 31 mice treated with a three-stage carcinogenesisprotocol (initiation with DMBA and promotion for 10 weeks withTPA followed by 10 weeks of benzoyl peroxide) gave rise to sixspindle cell carcinomas (0.19/mouse). The number of keratin-positivetumor cells and the intensity of the inununo-stain varied markedly,but all tumors expressed the 55 kd polypeptide. Although othercarcinogens, mainly UV radiation, have been able to induce spindlecell tumors, the present data indicate that chemical carcinogenesisprotocols are able to induce the formation of this highly malignantvariant of skin carcinoma.     

New strains of inbred SENCAR mice with increased susceptibility to induction of papillomas and squamous cell carcinomas in skin

To develop mouse strains useful for studies of susceptibility and resistance to the induction of skin tumors, three new inbred SENCAR strains were independently derived by random inbreeding of outbred SENCAR mice. Characterization of these mice for sensitivity to skin tumor development indicated that mice of all three strains displayed increased sensitivity to initiation by 7,12-dimethylbenz[a]anthracene (DMBA), urethane, or N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA). Promotion by mezerein as well as carcinogenesis by repeated treatment with DMBA or MNNG produced papillomas with a high frequency of conversion to squamous cell carcinomas (SCCs). Compared with outbred SENCAR mice, development of both squamous papillomas and carcinomas was increased at least two-fold by all protocols tested. The F₁ hybrid between SENCARA/Pt males and resistant BALB/cAnPt females was resistant to the induction of both papillomas and SCCs after initiation by 2 μg of DMBA and promotion by 20 weekly applications of 2 μg of TPA. Papillomas developed in all of the SENCARA/Pt mice, none of the BALB/cAnPt mice, and 12% of the F₁ progeny. Thus, at these doses of initiator and promoter, resistance was incompletely dominant in the F₁ hybrid. However, the responsiveness of the F₁ mice could be increased substantially by increasing the dose of the promoter. Mol. Carcinog. 20:143–150, 1997. © 1997 Wiley-Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.     

Keratin modifications in epidermis, papillomas, and carcinomas during two-stage carcinogenesis in the SENCAR mouse

To elucidate the role of keratin modification in tumor promotion, we investigated the keratin polypeptide patterns of mouse epidermis, papillomas, and carcinomas throughout an initiation-promotion experiment. The epidermal keratin modifications induced by repetitive 12-O-tetradecanoylphorbol-13-acetate treatments in both initiated and noninitiated mouse skin were essentially identical to those observed with a single 12-O-tetradecanoylphorbol-13-acetate application. These changes were even more pronounced in epidermal papillomas. In addition, the keratins of the papillomas displayed greater charge heterogeneity, particularly among the high-molecular-weight keratins (Mr 60,000 to 62,000). As the experiment progressed, there appeared to be a selective loss of one group of high-molecular-weight keratins (Mr 62,000) in some of the papillomas. Interestingly, the carcinomas that appeared at this time had significant reduction in both groups of high-molecular-weight keratins. In fact, the keratin profiles of carcinomas were very similar to the patterns observed in basal cells after a single 12-O-tetradecanoylphorbol-13-acetate treatment of adult epidermis. This may indicate that the program of keratin expression of a carcinoma becomes permanently fixed at a basal cell pattern. Changes in keratin patterns may serve as a biochemical marker of malignant progression in mouse epidermis.     

Sequential study of gamma-glutamyltransferase during complete and two-stage mouse skin carcinogenesis

The histochemical pattern of gamma-glutamyltransferase (GGT) was studied in benign and malignant tumors produced by two different experimental protocols, two-stage carcinogenesis and complete carcinogenesis. Six percent of all papillomas produced by two-stage carcinogenesis were GGT positive, whereas 14% of benign tumors produced by complete carcinogenesis exhibited GGT-positive areas. The incidence of GGT-positive papillomas in the two-step carcinogenesis protocol increased up to wk 28 of treatment. After 32 wk, the incidence decreased abruptly, coinciding with an abrupt increase in the incidence of squamous cell carcinomas. On the other hand, the incidence of GGT-positive benign tumors produced during the course of complete carcinogenesis increased gradually up to wk 32 of treatment, coinciding with the increased incidence of squamous cell carcinomas. The incidence of GGT-positive keratoacanthomas and GGT-positive papillomas produced with the complete carcinogenesis protocol exhibited different patterns, suggesting different histogenesis and biological behavior of these two types of tumors. In addition, the labeling index of GGT-positive areas was lower (17 +/- 3%) than that of the GGT-negative areas (41 +/- 0.18%) of the same papillomas, indicating that the presence of GGT may be related to abnormal keratocyte differentiation rather than to proliferative changes.     

Lack of concordant p53 mutations in some paired primary and metastatic mouse squamous cell carcinomas induced by chemical carcinogenesis

We studied the frequency and pattern of p53 mutations in 16 mouse skin primary squamous carcinomas induced by chemical carcinogens and their 19 matched metastases. The molecular changes were analyzed by polymerase chain reaction-single-strand conformation polymorphism and subsequent direct sequencing analysis. Eleven mutations of the p53 gene were detected in a total of eight primary tumors, and 10 mutations were detected in nine metastases. Only four pairs had identical mutations in primary and paired metastatic tumors. Eight mutations in six pairs were detected in primary tumors but not in their metastases, and four mutations from three matched pairs were detected in metastases but not in primary tumors. The four pairs that contained the same mutations in both the primary and secondary tumors had lymph-node metastases, and all mutations occurred in exon 8. Conversely, five of six pairs with p53 mutations only in primary tumors had lung metastases and only one of the mutations occurred in exon 8. None of the mutations found only in metastases were located in exon 8. These data indicate that p53 mutations are prevalent in lymph-node metastases and infrequent in lung metastases of mouse skin tumors and that primary tumors with exon 8 mutations may be more likely to metastasize to the lymph nodes. © 1995 Wiley-Liss Inc.     

SAGE profiling of UV-induced mouse skin squamous cell carcinomas, comparison with acute UV irradiation effects

Ultraviolet (UV) irradiation is the primary environmental insult responsible for the development of most common skin cancers. To better understand the multiple molecular events that contribute to the development of UV-induced skin cancer, in a first study, serial analysis of gene expression (SAGE) was used to compare the global gene expression profiles of normal SKH-1 mice epidermis with that of UV-induced squamous cell carcinomas (SCCs) from SKH-1 mice. More than 200 genes were found to be differentially expressed in SCCs compared to normal skin (P < 0.0005 level of significance). As expected, genes related to epidermal proliferation and differentiation were deregulated in SCCs relative to normal skin. However, various novel genes, not previously associated with skin carcinogenesis, were also identified as deregulated in SCCs. Northern blot analyses on various selected genes validated the SAGE findings: caspase-14 (reduced 8.5-fold in SCCs); cathepsins D and S (reduced 3-fold and increased 11.3-fold, respectively, in SCCs); decorin, glutathione S-transferase omega-1, hypoxia-inducible factor 1 alpha, insulin-like growth factor binding protein-7, and matrix metalloproteinase-13 (increased 18-, 12-, 12-, 18.3-, and 11-folds, respectively, in SCCs). Chemokine (C-C motif), ligand 27 (CCL27), which was found downregulated 12.7-fold in SCCs by SAGE, was also observed to be strongly downregulated 6-24 h after a single and multiple UV treatments. In a second independent study we compared the expression profile of UV-irradiated versus sham-treated SKH-1 epidermis. Interestingly, numerous genes determined to be deregulated 8 h after a single UV dose were also deregulated in SCCs. For instance, genes whose expression was upregulated both after acute UV-treated skin and SCCs included keratins 6 and 16, small proline-rich proteins, and S100 calcium binding protein A9. Studies like those described here do not only provide insights into genes and pathways involved in skin carcinogenesis but also allow us to identify early UV irradiation deregulated surrogate biomarkers of potential use in chemoprevention studies.     

Retinoyl-phorbol-acetate is a complete skin tumor promoter in SENCAR mice

The semi-synthetic phorbol ester 12-O-retinoyl phorbol 13-acetate (RPA) was tested for activity as a complete tumor promoter, as well as for first or second stage promotion activity, in the skin of SENCAR mice. Unlike in NMRI mice where RPA has been reported to act only as a second stage promoter, in SENCAR mice it has moderate complete promoting activity as well as second stage activity. RPA also induces epidermal hyperplasia and an increase in the number of dark basal keratinocytes.     

Absence of papillomavirus in skin tumors induced in SENCAR mice by a two-stage carcinogenesis protocol

SENCAR mice are unusually sensitive to induction of papillomas and squamous cell carcinomas by initiation with 7,12-dimethylbenzanthracene (DMBA) and promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA). Tumors induced by this protocol were tested for the presence of papillomavirus by immunohistochemistry, Southern blot, reverse Southern blot and dot blot hybridization techniques. Papillomavirus antigens were not detected in any of 235 tumors or 142 non-tumor-bearing skin samples analyzed. Southern blots and dot blots, using a mixed probe of cloned rodent papillomavirus DNA from the multimammate rat, Mastomys natalensis, and the European harvest mouse, Micromys minutus, did not reveal the presence of either episomal or integrated papillomavirus genomes in total cellular DNA extracts from the tumors or non-tumor-bearing skin. To circumvent the possibility that insufficient cross-homology existed to detect a papillomavirus genome with the mixed probe used, DNAs extracted from six papillomas were labeled and each used to probe reference blots that contained 25 cloned papillomavirus genomes excised from their vectors. No evidence for the presence of a papillomavirus genome was detected by this method. Therefore, it is unlikely that papillomaviruses play a role in the induction of tumors in SENCAR mice by two-stage carcinogenesis protocols.     

SENCAR mouse skin tumors produced by promotion alone have A to G mutations in codon 61 of the c-rasHa gene

SENCAR mice, developed by selective breeding for high susceptibilityto skin carcinogenesis by initiation with 7, 12-dimethylbenz[a]anthraceneand promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA),form squamous papillomas in     

Influence of diet and calorie restriction on the initiation and promotion of skin carcinogenesis in the SENCAR mouse model

Diets were restricted to 60% of the intake of the control mice by feeding less diet (total diet restriction, TDR) or by feeding fewer calories from fat and carbohydrate (calorie restriction, CR) during the initiation or promotion phases of skin tumorigenesis in female SENCAR mice. Skin cancer was initiated by topical treatment with 10 nmol of 7,12-dimethylbenzanthracene in acetone and promoted by twice weekly treatments with 12-O-tetradecanoylphorbol-13-acetate in acetone for 20 wk. Dietary restriction preceding and during 7,12-dimethylbenzanthracene treatment did not influence skin papilloma or carcinoma yield. Papilloma incidence and the number of papillomas per effective mouse were reduced in mice restricted by both TDR and CR protocols during and following promotion with 12-O-tetradecanoylphorbol-13-acetate. Papilloma size was reduced at experimental wk 16 and 20 in both TDR and CR groups fed these diet regimens during promotion. However, by wk 28 and 32, papilloma sizes were similar in the control and TDR groups, and smaller papillomas were observed only in the CR group. The average carcinoma latency was extended by 26% in the groups restricted during promotion, and incidence was reduced in both groups. The reduction, however, was statistically significant only in the CR group. Body weight gain was reduced during the times when dietary restriction was enforced, and in a short-term study, both restricted diet treatments reduced the percentage of carcass protein.     

Angiogenic switch occurs late in squamous cell carcinomas of human skin

Angiogenesis is a crucial event in carcinogenesis and its onset has been associated with premalignant tumour stages. In order to elucidate the significance of angiogenesis in different stages of epithelial skin tumours, we analysed the vessel density in ten normal skin samples, 14 actinic keratosis (AK), 12 hypertrophic AKs, and in nine early- and 16 late-stage squamous cell carcinomas (SCCs). Mean vascular density was quantitated by counting the number of CD 31-immunostained blood vessels and by morphometric assessment of stained vessel area by computer-assisted image analysis. The results from both methods were well correlated. Mean vascular density was similar in normal dermis and in AK, and only slightly elevated in hypertrophic AKs and early SCC stages (tumour thickness < 2 mm). Only late-stage SCCs infiltrating the subcutis exhibited a significant increase in vascularization. Vessel density was independent of tumour localization, degree of proliferation and inflammatory cell infiltration. Furthermore, tumour vascularization was not correlated with the expression of vascular endothelial growth factor, a major angiogenic factor, as revealed by in situ hybridization and immunohistochemistry. The restriction of enhanced vascularization to increased tumour thickness may be a major reason for the rather low metastatic spread of cutaneous SCCs.     

Effects of a biomimetic superoxide dismutase on complete and multistage carcinogenesis in mouse skin

The effects of a selective detoxifier of the proximate oxygenradical, superoxide anion, on the induction of tumors in theskin of CD-1 mice by either the initiation-promotion regimenor the complete carcinogenesis process were investigated. Theprinciple agent of interest, copper (II) (3,5-diisopropylsalicylate)₂(CuDIPS), is a low molecular weight, lipophilic copper coordinationcomplex that catalytically disproportionates superoxide anionat a rate comparable to native CuZn superoxide dismutase (SOD).The protocols used to elicit tumors were: (i) a single applicationof 0.2 µmol of 7,12-dimethylbenz[a]anthracene (DMBA) followedby twice-weekly applications of 12-O-tetradecanoylphorbol-13-acetate(TPA) in an initiation-promotion study, and (ii) either a singleapplication of 3.6 µmol DMBA followed by no further treatmentor weekly applications of 0.2 µmol DMBA in complete carcinogenesisprotocols. Application of 2 µmol CuDIPS 15 min prior tothe initiating dose of DMBA was without significant effect ontumor yield or incidence, whereas application prior to eachdose of TPA substantially reduced tumor incidence and yield.This anti-promoting property of CuDIPS can be attributed toits SOD-mimetic activity in as much as the corresponding zinccoordination complex lacking in SOD activity, zinc (II) (3,5-diisopropylsalicylate)₂,was non-inhibitory. Significant reductions in tumor yield werealso observed when CuDIPS was applied prior to DMBA in eitherof the complete carcinogenesis protocols. Additionally, covalentbinding of [³H]DMBA to epidermal DNA was markedly reduced byCuDIPS pre-treatment, suggesting that the anti-carcinogenicproperties may reflect a perturbation in superoxide anion-dependentmetabolic activation of DMBA. The induction by DMBA of ornithinedecarboxylase activity, a biochemical marker of tumor promoteractivity, was not affected by CuDIPS; however, induction ofornithine decarboxylase by TPA was potently blocked. Collectively,these effects of a biomimetic SOD further implicate reactiveoxygen species at multiple stages in chemical carcinogenesis.     

Initiation, promotion and complete carcinogenesis by N-methyl-N'-nitro-N-nitrosoguanidine or ethylnitrosourea in the Sencar mouse skin tumorigenesis model

Five doses of either N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or ethylnitrosourea (ENU) were tested as complete carcinogens, tumor initiators and tumor promoters in the SENCAR skin tumorigenesis model. As tumor initiators, MNNG-induced papillomas at all doses tested, while ENU was active from 10-40 mumol. As complete carcinogens, MNNG from doses of 0.5-5.0 mumol and ENU from doses of 10 mumol-40 mumol were potent inducers of both papillomas and carcinomas indicating that these agents are active as both tumor initiators and tumor promoters.     

Transgenic mice and squamous multistage skin carcinogenesis

The use of animals models of human cancers has proved useful in the elucidation of molecular events which occur during tumour development. Mouse skin has been used as a model for human squamous cancer for a number of decades, and analysis of this model has identified a number of changes important for the evolution of malignancy. Transgenic mice offer a further avenue of advancement, allowing refinement of the model, and the ability to examine the consequences of individual eventsin vivo in greater detail. This article reviews the impact of transgenic approaches to our understanding of multistage squamous carcinogenesis in mouse skin.     

The role of UV-B light in skin carcinogenesis through the analysis of p53 mutations in squamous cell carcinomas of hairless mice

Mutation spectra of the p53 gene from human skin carcinomas have been connected to solar UV radiation. For comparison we have characterized the mutation spectrum of the p53 gene in a very large sample of squamous cell carcinomas from hairless mice induced with UV of wavelength 280-320 nm (UV-B), which have substantiated the mutagenic effects of UV-B radiation in vivo. Tumors from hairless mice, random bred SKH:HR1 as well as inbred SKH:HRA strains, which are analyzed for mutations in the conserved domains of the p53 protein present a very specific mutation spectrum. The observed mutation frequency after chronic UV-B radiation in the p53 gene ranged from 54% (SKH-HRA) to 73% (SKH-HR1) among the 160 tumors analyzed. Over 95% of the mutations were found at dipyrimidine sites located in the non-transcribed strand, the majority were C-->T transitions and 5% were CC-->TT tandem double mutations. Four distinct UV-B mutation hot spots have been identified for the first time: two major ones at codons 267 (33%) and 272 (19%) and two minor ones at codons 146 (10%) and 173 (4%). The codon 267 hot spot consists of a CpG preceded by a pyrimidine, which confirms in vivo an important role for this UV-B mutable site in UV-B-induced skin tumors that is not found in other types of mouse tumors. Comparison with mutation spectra from human skin carcinomas fully validates the merits of the hairless mouse model for studying the molecular mechanisms of skin carcinogenesis. For example, the murine hot spot at codon 272 does have a full equivalent in human skin carcinomas. In contrast, the human equivalent of the murine codon 267 lacks the dipyrimidine site and therefore fails to be a pronounced hot spot in human skin carcinomas; however, this site is one of the major hot spots in human internal cancers (evidently not induced by UV radiation but probably by deamination of the 5 methyl cytosine).      

Carcinogen-specific mutational pattern in the p53 gene in ultraviolet B radiation-induced squamous cell carcinomas of mouse skin.

We have examined 35 epidermal tumors induced in mice of four different strains by chronic exposure to ultraviolet B radiation for the presence of aberrations in the p53 tumor suppressor gene. Polymerase chain reaction products from p53 exons 5 to 8 were screened by single-strand conformation polymorphism analysis and sequencing. Base substitutions were found in seven tumors (20%). All mutations occurred at dipyrimidine sequences; most frequent were C-->T single base and CC-->TT tandem transitions suggesting the involvement of UV radiation in the genesis of the mutations. Three base substitutions were located at codon 148, and all dipyrimidine-derived mutations occurred at sites where the sequence is present in the nontranscribed DNA strand, indicating some site and strand specificity of the ultraviolet B-induced p53 mutations.