Skin Tumor Development In Mice Research Articles

E2F4 and E2F1 Have Similar Proliferative Properties but Different Apoptotic and Oncogenic Properties In Vivo

Loss of retinoblastoma (Rb) tumor suppressor function, as occurs in many cancers, leads to uncontrolled proliferation, an increased propensity to undergo apoptosis, and tumorigenesis. Rb negatively regulates multiple E2F transcription factors, but the role of the different E2F family members in manifesting the cellular response to Rb inactivation is unclear. To study the effect of deregulated E2F4 activity on cell growth control and tumorigenesis, transgenic mouse lines expressing the E2F4 gene under the control of a keratin 5 (K5) promoter were developed, and their phenotypes were compared to those of previously generated K5 E2F1 transgenic mice. In contrast to what has been observed in vitro, ectopically expressed E2F4 was found to localize to the nucleus and induce proliferation to an extent similar to that induced by E2F1 in transgenic tissue. Unlike E2F1, E2F4 does not induce apoptosis, and this correlates with the differential abilities of these two E2F species to stimulatep19ARF expression in vivo. To examine the role of E2F4 in tumor development, the mouse skin two-stage carcinogenesis model was utilized. Unlike E2F1 transgenic mice, E2F4 transgenic mice developed skin tumors with a decreased latency and increased incidence compared to those characteristics in wild-type controls. These findings demonstrate that while the effects of E2F1 and E2F4 on cell proliferation in vivo are similar, their apoptotic and oncogenic properties are quite different.

Ras activity and cyclin D1 expression: An essential mechanism of mouse skin tumor development

ras activity and cyclin D1 expression: An essential mechanism of mouse skin tumor development

Ras activity and cyclin D1 expression: An essential mechanism of mouse skin tumor development

ras is a family of small GTP-binding proteins that transduce signals from tyrosine-kinase receptors to the nucleus and thus play a role in the regulation of cell proliferation and differentiation. Several lines of evidence have shown that the cell-cycle machinery, specifically the circuit cyclin D1/cyclin-dependent kinase (cdk) 4 and 6-p16-pRb, lies downstream of ras. Point mutations that activate the ras protein and its downstream cascade have been observed in human and experimental tumors. In particular, ras mutations have been well characterized in the mouse skin two-stage carcinogenesis model, and a large body of literature has indicated that initiation with the genotoxic carcinogen 7,12-dimethylbenz[a]anthracene induces a specific point mutation in Ha-ras gene in this model. In the last few years, several studies have shown a correlation between ras activation and alterations in the expression of cyclin D1 as well as other cell cycle-regulated proteins, but the actual role of these alterations in tumor development had not been determined until a recent study provided genetic and biochemical evidence that cyclin D1 is a critical target of oncogenic ras in mouse skin carcinogenesis. Here we review these results, including the evidence that cyclin D1 has a role as a downstream mediator of ras activity during tumor development. We propose a model in which cyclin D1 has a unique growth-promoting role in tumor development but does not act as an oncogene independently of ras activity.

Arsenic Enhancement of Skin Neoplasia by Chronic Stimulation of Growth Factors

Although numerous epidemiological studies have shown that inorganic arsenicals cause skin cancers and hyperkeratoses in humans, there are currently no established mechanisms for their action or animal models. Previous studies in our laboratory using primary human keratinocyte cultures demonstrated that micromolar concentrations of inorganic arsenite increased cell proliferation via the production of keratinocyte-derived growth factors. As recent reports demonstrate that overexpression of keratinocyte-derived growth factors, such as transforming growth factor (TGF)-alpha, promote the formation of skin tumors, we hypothesized that similar events may be responsible for those associated with arsenic skin diseases. Thus, the influence of arsenic in humans with arsenic skin disease and on mouse skin tumor development in transgenic mice was studied. After low-dose application of tetradecanoyl phorbol acetate (TPA), a marked increase in the number of skin papillomas occurred in Tg.AC mice, which carry the v-Ha-ras oncogene, that received arsenic in the drinking water as compared with control drinking water, whereas no papillomas developed in arsenic-treated transgenic mice that did not receive TPA or arsenic/TPA-treated wild-type FVB/N mice. Consistent with earlier in vitro findings, increases in granulocyte/macrophage colony-stimulating factor (GM-CSF) and TGF-alpha mRNA transcripts were found in the epidermis at clinically normal sites within 10 weeks after arsenic treatment. Immunohistochemical staining localized TGF-alpha overexpression to the hair follicles. Injection of neutralizing antibodies to GM-CSF after TPA application reduced the number of papillomas in Tg.AC mice. Analysis of gene expression in samples of skin lesions obtained from humans chronically exposed to arsenic via their drinking water also showed similar alterations in growth factor expression. Although confirmation will be required in nontransgenic mice, these results suggest that arsenic enhances development of skin neoplasias via the chronic stimulation of keratinocyte-derived growth factors and may be a rare example of a chemical carcinogen that acts as a co-promoter.

Induction of apoptosis by an inhibitor of cAMP-specific PDE in malignant murine carcinoma cells overexpressing PDE activity in comparison to their nonmalignant counterparts

In order to study potential changes in phosphodiesterase (PDE) activity associated with malignant transformation, normal primary keratinocytes and cells corresponding to different stages of epidermal tumor development in mouse skin were analyzed with respect to their 3',5'-cyclic adenosine monophosphate (cAMP) hydrolyzing activity. Expression of cAMP-specific PDE-4, intracellular cAMP content, and the sensitivity to the growth inhibitory effect of the PDE-4-specific inhibitor 7-benzylamino-6-chloro-2 piperazino-4-pyrrolidino-pteridine (DC-TA-46) were studied in the two papilloma cell lines, MSCP6 and 308, and in the highly malignant carcinoma cell line CarB. No significant difference in soluble PDE activity and in intracellular cAMP was found in the two papilloma cell lines when compared to primary keratinocytes. In contrast, the spindle-cell carcinoma cell line CarB exhibited significantly higher PDE activity, concomitant with the lowest cAMP level. In all cell lines and also in the primary keratinocytes, rolipram-sensitive PDE-4 activity accounted for the major cAMP-hydrolyzing activity. In primary keratinocytes and in MSCP6 cells, the PDE-4 inhibitor DC-TA-46 induced at best marginal growth inhibition, whereas cell growth of 308 cells was markedly affected at concentrations > 2 microM. The carcinoma cell line CarB showed the highest sensitivity to DC-TA-46 (IC50 = 0.8 +/- 0.3 microM). Treatment of CarB cells with DC-TA-46 strongly inhibits intracellular PDE activity, resulting in a marked and long-lasting rise of cAMP. After 24 h of treatment, arrest in the G0/G1 phase of the cell cycle is induced. Treatment with concentrations > 2 microM of this highly effective PDE inhibitor results in induction of apoptotic cell death, as detected by fluorescence microscopy, flow cytometry, and ELISA-based determination of fragmented DNA in intact cells.

Arsenic can mediate skin neoplasia by chronic stimulation of keratinocyte-derived growth factors

Although numerous epidemiological studies have shown that inorganic arsenicals are human skin carcinogens, there is currently no accepted mechanism for its action or an established animal model for its study. We observed increased mRNA transcripts and secretion of keratinocyte growth factors, including granulocyte macrophage-colony stimulating factor (GM-CSF) and transforming growth factor-alpha (TGF-alpha) and the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) in primary human epidermal keratinocytes cultured in the presence of low micromolar concentrations of sodium arsenite. Total cell numbers, as well as c-myc expression and incorporation of [3H]thymidine, both indicators of cell proliferation, were also elevated in keratinocyte cultures treated with sodium arsenite. As an in vivo model, the influence of arsenic on mouse skin tumor development was studied in transgenic TG.AC mice which carry the v-Ha-ras oncogene, and can serve as a genetically initiated model for skin carcinogenesis. Following low-dose application of 12-O-tetradecanoyl phorbol-13-acetate (TPA), a marked increase in the number of skin papillomas occurred in transgenic mice receiving arsenic in the drinking water as compared to control drinking water. Papillomas did not develop in arsenic-treated transgenic mice that had not received TPA or arsenic-treated wild-type FVB/N mice, suggesting that arsenic is neither a tumor initiator or promoter but rather an enhancer. Injection of anti-GM-CSF antibodies following application of TPA in transgenic mice reduced the number of papillomas. Consistent with that observed in human keratinocyte cultures, increases in GM-CSF and TGF-alpha mRNA transcripts were found within the epidermis of arsenic-treated mice when compared to controls within 6 weeks of treatment. These results suggest that arsenic enhances papilloma development via the chronic stimulation of keratinocyte-derived growth factors and represents the first example of a chemical carcinogen that acts in this manner. These studies suggest that in vitro studies with human keratinocyte cultures examined in conjunction with TG.AC transgenic mice can provide a useful model for examining the tumor enhancing properties of environmental chemicals.

Purification and characterization of the epidermal 8(S)lipoxygenase.

Skin epidermis forms a vast source of arachidonic acid metabolites. In cells, arachidonic acid is esterified to phopspholipids. Once released from its intracellular stores, arachidonic acid serves as a substrate for different families of oxygenases such as the prostaglandinsynthases, the cytochrome P-450-dependent epoxygenases and the lipoxygenases. Regulation of eicosanoid biosynthesis occurs both at the level of arachidonic acid mobilization and conversion into eicosanoids (for a review see [1]). Eicosanoids play a critical role in skin tumor development in mice. A striking event seen among the multiple changes in epidermal arachidonic acid metbolism produced by the tumor-promoting phorbol ester TPA is the induction ofa cytosolic 8(S)-lipoxygenase (8-LOX) activity. The enzyme catayzes the conversion of free arachidonic acid to 8(S)-HPETE and of free linoleic acid to 9(S)-HPODE. 8-LOX inducton is restricted to the suprabasal compartment of the epidermis and depends on transcriptional activation and de novo protein synthesis. A constitutive overexpression of 8-LOX activity has been observed in papillomas (2).

Arachidonate Has Protumor-Promoting Action that is Inhibited by Linoleate in Mouse Skin Carcinogenesis

Previous studies demonstrated a requirement for arachidonic acid metabolites in tumor development in mouse skin. The goal of this study was to determine whether the arachidonate content of epidermal phospholipids could be altered by increasing dietary levels of linoleate and whether specific metabolites of linoleate and arachidonate have dissimilar biological effects. In a series of tumor studies in which the quantity of dietary linoleate was incrementally increased, a slight reduction in phospholipid levels of arachidonate was observed that correlated with an increased phospholipid level of linoleate and a suppression in tumor yield. A comparison of the arachidonate lipoxygenase metabolite 12-hydroxyeicosatetraenoic acid (12-HETE) with the 13-hydroxyoctadecadienoic acid (13-HODE) lipoxygenase metabolite of linoleate revealed that 12-HETE has biological activities that mimic the phorbol ester tumor promoters, whereas 13-HODE has antithetical effects. Specifically, 12(S)-HETE enhanced the activation of protein kinase C by phorbol esters, mimicked phorbol ester-induced adhesion of keratinocytes to fibronectin and mimicked phorbol ester repression of expression of a differentiation-related gene, keratin-1. 13-HODE blocked 12-HETE-induced cell adhesion and prevented 12-HETE-induced suppression of keratin-1 expression. Overall, these studies suggest that arachidonate and linoleate have opposing functions in the epidermis, particularly with regard to events involved in tumor development.

12-Lipoxygenase isoenzymes in mouse skin tumor development.

12-lipoxygenase-catalyzed arachidonic acid metabolism in normal and neoplastic mouse epidermis was assessed by cDNA cloning of the epidermal 12-lipoxygenases and by studying their expression patterns, enzyme activities, and product levels. Papillomas and squamous cell carcinomas induced by the initiation/promotion protocol contained 50- to 60-fold more 12-hydroxy-5,8,10,14-eicosatetraenoic acid (HETE) than normal epidermis. The ratio of S to R enantiomers was 9:1. This indicates that most of this eicosanoid was of enzymatic origin. Accordingly, cell-free preparations of the tumors exhibited about fivefold elevated 12-lipoxygenase activities. A papilloma-derived cDNA library was screened with human platelet-type 12-lipoxygenase cDNA probes. Two cDNA clones encoding the platelet-type and the leukocyte-type isoforms of murine 12-lipoxygenase were isolated, demonstrating the coexpression of the isoenzymes in the same tissue and species. When expressed in COS-7 cells, the recombinant enzymes showed the characteristic substrate selectivity and product profile, with the leukocyte-type enzyme metabolizing linoleic and arachidonic acid to 13-hydroxy-9,11-octadecadienoic acid and to 12- and 15-HETE, respectively, and the platelet-type enzyme oxygenating exclusively arachidonic acid to 12-HETE. In epidermis in vivo and in keratinocytes in culture, only the platelet-type 12-lipoxygenase (mRNA and protein) was detectable. In mouse epidermis both isoenzymes were induced transiently by phorbol esters. Most tumors showed constitutive overexpression of platelet-type mRNA, whereas leukocyte-type specific transcripts were detectable only in a few tumors. These data suggest that the platelet-type enzyme is the 12-lipoxygenase isoform of keratinocytes that is responsible for the generation of most of the 12-HETE found in neoplastic epidermis.

Differential expression of prostaglandin H synthase isozymes during multistage carcinogenesis in mouse epidermis.

An anti-tumor-promoting effect of indomethacin and related nonsteroidal anti-inflammatory drugs (NSAIDs) as well as the ability of the tumor promoter 12-O-tetradecanoylphorbol-13- acetate (TPA) to increase the level of prostaglandins in murine keratinocytes and mouse epidermis in vivo has been repeatedly documented. Here, the expression of prostaglandin H synthase (PGHS) isozymes, which are major targets of NSAIDs, was investigated in different stages of tumor development in mouse skin. Mouse epidermis in vivo constitutively expressed PGHS-1. PGHS-1 steady-state levels remained unchanged upon induction of acute or chronic epidermal hyperplasia by TPA and in papillomas and carcinomas generated by the initiation-promotion procedure, with 7,12-dimethylbenz[a]anthracene as initiator and TPA as promoter. Thus, the elevated prostaglandin level in the acute hyperplastic epidermis was very likely due to PGHS-2 induction. Repeated applications of TPA resulted in stationary hyperplasia and downregulation of PGHS-2 expression and prostaglandin levels, suggesting that the epidermis had adapted to the TPA stimulus. In papillomas and carcinomas, however, constitutive overexpression of PGHS-2 was found, with a large amount of prostaglandin E2 and prostaglandin F2 alpha. Keratinocyte cell lines corresponding to different stages of tumor development also constitutively over-expressed PGHS-2. Considered with inhibitor studies, these data suggest that PGHS-2 has a critical role in skin carcinogenesis. The anti-tumor-promoting effect of the PGHS inhibitor indomethacin is specifically reversed by prostaglandin F2 alpha, indicating that this prostaglandin type has a significant role in tumor development.

Transcription factors junB and c-jun are selectively up-regulated and functionally implicated in fibrosarcoma development.

Bovine papillomavirus transgenic mice develop skin tumors arising from dermal fibroblasts in a process comprised of three distinctive stages: mild and aggressive fibromatoses, and fibrosarcoma. In both tissue biopsies and derivative cell lines, the proto-oncogenes junB and c-jun are induced in the latter two stages, in contrast to junD and fos. Fibrosarcoma cell lines have increased AP-1 DNA-binding activity. Overexpression of junB or c-jun by transfection into the mild fibromatosis stage elicited changes in cell shape and anchorage independence, whereas junD did not. Similar transfections of normal skin fibroblasts had no effect. Thus, junB and c-jun represent progression factors whose activities are necessary at an intermediate stage of tumor development, subsequent to the initiation of aberrant proliferation.

Chemoprevention of chemically induced skin tumor development by diallyl sulfide and diallyl disulfide.

Garlic and onion oil have been shown to inhibit chemically induced skin tumor development in mice. In the present study, the effects of diallyl sulfide and diallyl disulfide, oil-soluble constituents of garlic and onion, on 7,12-dimethylbenz(a)anthracene-induced and 12,O-tetradecanoylphorbol-13-acetate-promoted skin tumor formation were examined in SENCAR mice. Topical application of diallyl sulfide or diallyl disulfide significantly inhibited skin papilloma formation from the ninth week of promotion and significantly increased the rate of survival in the murine model. Our findings support earlier evidence that these naturally occurring compounds may be useful for the chemoprevention of certain types of tumors.

Differences in skin carcinogenesis by methylnitrosourea between mice of several strains

To compare the susceptibilities of the skin of different strains of mice to the carcinogenic effect of a directly acting alkylating agent, groups of 20 mice were treated twice a week with 25 μl of a solution of methylnitrosourea in methanol. The solution was 0.04M and was applied to the shaved back of female BALB c . Sencar, CD-1 and Swiss mice for 25 weeks. Four groups of 20 mice of each strain were 8 weeks old at the beginning of treatment. Another four groups were 58 weeks old when treatment began. More of the BALB c mice developed skin tumors than the other three strains, the Sencar mice somewhat less. Few CD-1 mice developed skin tumors and about one third of the Swiss mice. In all four strains, there were fewer animals with skin tumors among those begun at 58 weeks than in the young mice, but the difference was small. Survival was poor among CD-1 mice, but there was not a large difference between the strains in time of appearance of first tumor, or in average latent period of skin tumors, almost all of which were carcinomas. The Sencar mice were not out-standingly more sensitive to skin carcinogenesis by MNU, as they were to UV radiation-induced skin carcinogenesis. In a comparable study in Swiss mice neither dimethylnitrosourea nor diethylnitrosourea induced skin tumors by painting and both showed only a weak systemic carcinogenic effect in the lungs, although they are directly acting mutagens.

Genetic changes in skin tumor progression: Correlation between presence of a mutant ras gene and loss of heterozygosity on mouse chromosome 7

Initiation of tumorigenesis in mouse skin can be accomplished by mutagenesis of the H- ras gene by treatment with chemical carcinogens. A mouse model system has been developed to study the additional genetic events that take place during tumor progression. Skin carcinomas were induced in F1 hybrid mice exhibiting restriction fragment length polymorphisms at multiple chromosomal loci. Analysis of loss of heterozygosity in such tumors showed that imbalance of alleles on mouse chromosome 7, on which the H- ras gene is located, occurs very frequently in skin carcinomas. The chromosomal alterations detected, which included both nondisjunction and mitotic recombination events, were only seen in tumors that have activated ras genes. We conclude that gross chromosomal alterations that elevate the copy number of mutant H- ras and/or lead to loss of normal H- ras are a consistent feature of mouse skin tumor development.

Effects of immunosuppressive therapy on the induction of skin tumors by ultraviolet irradiation in hairless mice.

The effects of therapy with four commonly used immunosuppressants--azathioprine, prednisolone, cyclophosphamide, and cyclosporine, on (UVI)-induced skin carcinogenesis were studied in the albino hairless (HRA/Skh-1) mouse. Following 30 weeks' exposure to UVI (290-400 nm) alone, 87% of mice developed skin tumors; the mean incidence of tumors at that time was 2.4 per mouse; and the tumors were predominantly papillomas (72%), with the remainder being carcinomas (25%) and keratoacanthomas (3%). Mice received immunosuppressive drug therapy beginning shortly after the start of UVI and continuing for up to 28 weeks. All drugs were given at immunosuppressive levels and dosages were comparable on a body weight basis to those used in clinical transplantation. Prednisolone had no effect on UVI-induced tumor development. Cyclosporine caused a moderate reduction in the latent period for tumor induction. Azathioprine and cyclophosphamide had strong promoting effects; the latent period for tumor induction was shortened and the tumor yield per mouse was increased (4.3 and 5.7 tumors per mouse, respectively, at 30 weeks after the start of UVI). Azathioprine, but not cyclophosphamide, also induced a larger proportion of carcinomas (43% and 15%, respectively). The results suggest that for kidney transplant recipients treated with the standard immunosuppressive drug regimen of azathioprine/prednisone, the increased susceptibility of the sun-exposed skin of these patients to squamous cell carcinoma is likely to be contributed to by specific promotion by the azathioprine therapy of the carcinogenic effects of sunlight.

Aneuploidy, an early event in mouse skin tumor development.

It has been suggested that chromosomal alterations can play a role in mouse skin carcinogenesis. These changes have been proposed as a possible mechanism of action of tumor promoters and they have also been related to the conversion of benign papillomas to carcinomas. However, direct evidence showing the chromosomal constitution of these tumors has not been previously described. Here we show the presence of aneuploid cells in very early papillomas and the eventual displacement of the diploid stem line by aneuploid clones at later stages. Squamous cell carcinomas (SCC) induced by the same protocol were highly aneuploid. These results suggest that genomic imbalance produced by aneuploidy may be related to the malignant conversion of the benign papillomas.

Sunscreens for delay of ultraviolet induction of skin tumors

Sunscreens with different sun protection factors (SPFs) have been tested for their capability of delaying or preventing actinic damage and skin cancer development in groups of hairless, pigmented mice exposed to artificial ultraviolet (UV) light of increasing intensity. The dose delivered was less than or equal to 1 minimal erythema dose (MED) in the group of untreated mice, so that the mice to which sunscreens were applied never obtained a sunburn after UV exposure. The quality of UV light was similar to bright midday sun at a latitude of 56 degrees (city of Copenhagen). Tumorigenesis was demonstrated to be delayed corresponding to the SPF claimed by the manufacturer, but almost all of the UV-irradiated mice developed skin tumors. Histologic examination revealed actinic degeneration and tumors of squamous cell type with marked variation in differentiation. Metastases to lymph nodes and lungs were found in only 10%. Toxic reactions, such as eczematous-like skin reactions, dark coloring, and amyloidosis, were observed predominantly in the group treated with the sunscreen of highest SPF value. Long-term investigations seem to be necessary to unveil these problems--in particular, the specific SPF value, in sunscreens, that should be recommended to the public for prevention or delay of actinic damage and/or cancer development.

Mammary carcinoma in mice following the intranasal administration of methylcholanthrene

Experiments were conducted in 5 pure strains of mice (only the IF and CBA strains are important in this study as they do not naturally develop breast tumors) to study the histogenesis of mammary cancer. 0.5% methylcholanthrene was administered intranasally at 14-day intervals until the mice died. Of 29 IF females 18 developed mammary cancer the earliest tumor appearing after 4.4 months. The breast cancers were frequently multiple. Some of the tumors were tubular columnar- or cuboidal- celled carcinoma some were solid polyhedral-celled carcinoma and others were further dedifferentiated in places to spindle-celled carcinoma. There were 4 cases of squamous metaplasia; 6 had skin tumors. In 34 estrogen-treated males 11 developed mammary carcinoma the earliest tumor appearing after 5.4 months of treatment with methylcholanthrene. 7 mice developed skin tumors (5 malignant and 2 doubtful). The CBA mice exhibited less clear-cut results. Of the 22 females 4 showed macroscopic breast tumors; 1 of the 35 males had a mammary tumor. There were 20 cases of skin tumors (1 or 2 were malignant). The frequency of skin tumors was not significantly different in mice which developed mammary tumors.