The role of skin painting in predicting lung cancer.

The mouse skin cancer model provides an important system for studying mechanisms involved in the various stages of carcinogenesis and for bioassaying tobacco smoke constituents and additives for carcinogenic/cocarcinogenic and tumor-promoting properties as well as for identifying compounds that may inhibit tumor formation and malignant conversion. In addition, it is an excellent model for studying the formation of precancerous lesions as well as squamous cell carcinomas. It relates very well to other squamous cell carcinoma models and contributes to better understanding of the human epithelial cancers including lung cancer. The SENCAR mouse is an established model system demonstrated to be more sensitive than the B6C3F1 or Swiss CD-1 strains in the initiation/promotion skin-painting test method. Although the relationship between mouse skin tumors and any manifestation of the toxicity of tobacco smoke and other complex environmental mixtures in humans is unknown, the skin-painting model is the only assay that provides a practical method of obtaining a tumorigenic end point with cigarette smoke condensates and other complex mixtures. This assay provides a rapid response with relative ease of quantification of various parameters of tumorigenic response including tumor incidence, latency, multiplicity, and malignancy.

Avicins, a family of triterpenoid saponins from Acacia victoriae (Bentham), suppress H-ras mutations and aneuploidy in a murine skin carcinogenesis model.

We tested the ability of avicins, a family of triterpenoid saponins obtained from Acacia victoriae (Bentham) (Leguminosae: Mimosoideae), to inhibit chemically induced mouse skin carcinogenesis. Varying doses of avicins were applied to shaved dorsal skin of SENCAR mice 15 min before application of 100 nmol of 7,12-dimethylbenz[a]anthracene (DMBA) twice a week for 4 weeks (complete carcinogenesis model). The dorsal skin of a second group of mice was treated with one dose of 10 nmol of DMBA. Avicins were then applied 15 min before repetitive doses of 2 microg of phorbol 12-tetradecanoate 13-acetate (TPA) twice a week for 8 weeks (initiation/promotion model). At 12 weeks, avicins produced a 70% decrease in the number of mice with papillomas and a greater than 90% reduction in the number of papillomas per mouse in both protocols. We also observed a 62% and 74% reduction by avicins in H-ras mutations at codon 61 in the DMBA and DMBA/TPA models, respectively, as well as a significant inhibition of the modified DNA base formation (8-OH-dG) in both protocols. Marked suppression of aneuploidy occurred with treatment at 16 weeks in the initiation/promotion experiment. These findings, when combined with the proapoptotic property of these compounds and their ability to inhibit hydrogen peroxide (H(2)O(2)) generation, nuclear factor-kappaB (NF-kappaB) activation, and inducible nitric oxide synthase (iNOS) induction reported elsewhere, suggest that avicins could prove exciting in reducing oxidative and nitrosative stress and thereby suppressing the development of human skin cancer and other epithelial malignancies.

Altered skin development and impaired proliferative and inflammatory responses in transgenic mice overexpressing the glucocorticoid receptor.

Glucocorticoids (GCs) are potent inhibitors of epidermal proliferation and effective anti-inflammatory compounds, which make them the drug of choice for a wide range of inflammatory and hyperproliferative skin disorders. GC action is mediated via the glucocorticoid receptor (GR). To study the role of GR in skin development and the molecular mechanisms underlying its action, we generated transgenic mice overexpressing GR in epidermis and other stratified epithelia, under the control of the keratin K5 promoter. Newborn mice show altered skin development, manifested as variable-sized skin lesions that range from epidermal hypoplasia and underdeveloped dysplastic hair follicles to a complete absence of this tissue. In the most affected individuals, skin was absent at the cranial and umbilical regions, and the vibrissae and eyebrows appear scarce, short, and curly. In addition, as a consequence of transgene expression in other ectodermally derived epithelia, K5-GR mice exhibited further abnormalities that strikingly resemble the clinical findings in patients with ectodermal dysplasia, which includes aplasia cutis congenita. In adult transgenic skin, topical application of the tumor promoter TPA did not elicit hyperplasia or transcriptional induction of several proinflammatory cytokines. This anti-inflammatory role of GR was due at least in part to interference with NF-kB, leading to a strong reduction in the kB-binding activity without altering the transcriptional levels of the inhibitor IkBa.

2-methoxyestradiol blocks cell-cycle progression at G(2)/M phase and inhibits growth of human prostate cancer cells.

2-Methoxyestradiol (2-ME), an endogenous metabolite of 17beta-estradiol, is present in human blood and urine. Here we show for the first time that 2-ME significantly inhibited the growth of normal prostate epithelial cells and androgen-dependent LNCaP and androgen-independent DU145 prostate cancer cells. This growth inhibition was accompanied by a twofold increase in the G(2)/M population, with a concomitant decrease in the G(1) population, as shown by cell-cycle analysis. 2-ME treatment affected the cell-cycle progression of prostate cancer cells specifically by blocking cells in the G(2) phase. Immunoblot analysis of the key cell-cycle regulatory proteins in the G(2)/M phase showed a 14-fold increase in the expression of p21 and an eightfold increase in the expression of p34 cell division cycle 2 (cdc2). We also found an accumulation of phosphorylated cdc2 after 2-ME treatment. Furthermore, Wee 1 kinase was detectable after 2-ME treatment. 2-ME treatment also led to an increase in the activity of caspase-3, followed by apoptosis, as shown by terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate-biotin nick end-labeling and fluorescein isothiocyanate-poly(ADP-ribose) polymerase assay. Estrogen receptor levels did not change after treatment with 2-ME. Examination of the signaling pathways that mediate 2-ME-induced apoptosis showed reduction in the level of p53 expression and its DNA-binding activity. Given the fact that p53 mutations are common in patients with metastatic prostate cancer, our finding that 2-ME-mediated growth inhibition of human prostate cancer cells occurred in a p53-independent manner has considerable clinical significance. These findings, combined with the limited toxicity of 2-ME, may have significant implications for alternative treatment of advanced prostate cancer.

Induction of beta-transducin repeat-containing protein by JNK signaling and its role in the activation of NF-kappaB.

Activation of Jun N-kinase (JNK) and NF-kappaB transcription factor are the hallmarks of cellular response to stress. Phosphorylation of NF-kappaB inhibitor (IkappaB) by respective stress-inducible kinases (IKK) is a key event in NF-kappaB activation. beta-TrCP F-box protein mediates ubiquitination of phosphorylated IkappaB via recruitment of SCF(beta-TrCP)-Roc1 E3 ubiquitin ligase complex. Subsequent proteasome-dependent degradation of IkappaB results in activation of the NF-kappaB pathway. We found that a variety of cellular stress stimuli induce an increase in the steady state levels of beta-TrCP mRNA and protein levels in human cells. Activation of stress-activated protein kinases JNK (and, to a lesser extent, p38) by forced expression of constitutively active mutants of JNKK2 and MKK6 (but not MEK1 or IKKbeta) also leads to accumulation of beta-TrCP. Transcription of the beta-TrCP gene is not required for JNK-mediated induction of beta-TrCP. A synergistic effect of stimulation of IKK and JNK on the transcriptional activity of NF-kappaB was observed. The mechanisms of beta-TrCP induction via stress and its role in NF-kappaB activation are discussed.

Both (+/-)syn- and (+/-)anti-7,12-dimethylbenz[a]anthracene-3,4-diol-1,2-epoxides initiate tumors in mouse skin that possess -CAA- to -CTA- mutations at Codon 61 of c-H-ras.

We have determined the tumor-initiating activity of (+/-)syn- and (+/-)anti-7,12-dimethylbenz[a]anthracene-3,4-diol-1,2-epoxide (syn- and anti-DMBADE), the two metabolically formed bay-region diol epoxides of DMBA, and we have also analyzed mutations in the H-ras gene from tumors induced by these compounds. Using a two-stage, initiation-promotion protocol for tumorigenesis in mouse skin, we have found that both syn- and anti-DMBADE are active tumor initiators, and that the occurrence of papillomas is carcinogen dose dependent. All of the papillomas induced by syn-DMBADE (a total of 40 mice), 96% of those induced by anti-DMBADE (a total of 25 mice), and 94% of those induced by DMBA (a total of 16 mice) possessed a -CAA- to -CTA- mutation at codon 61 of H-ras. No mutations in codons 12 or 13 were detected in any tumor. Topical application of syn- and anti-DMBADE produced stable adducts in mouse epidermal DNA, most of which comigrated with stable DNA adducts formed after topical application of DMBA. Further analysis of the data showed that levels of the major syn- and anti-DMBADE-deoxyadenosine adducts formed after topical application of DMBA are sufficient to account for the tumor-initiating activity of this carcinogen on mouse skin. Previously, we showed that both the syn- and anti-DMBADE bind to the adenine (A182) at codon 61 of H-ras. Collectively, these results indicate that the adenine adducts induced by both bay-region diol epoxides of DMBA lead to the mutation at codon 61 of H-ras and, consequently, initiate tumorigenesis in mouse skin.

Wnt/beta-catenin signaling induces the expression and activity of betaTrCP ubiquitin ligase receptor.

Beta-transducing repeat-containing protein (betaTrCP) targets the ubiquitination and subsequent degradation of both beta-catenin and IkappaB, thereby playing an important role in beta-catenin/Tcf and NF-kappaB-dependent signaling. Here evidence is presented that beta-catenin/Tcf signaling elevates the expression of betaTrCP mRNA and protein in a Tcf-dependent manner, which does not require betaTrCP transcription. Induction of betaTrCP expression by the beta-catenin/Tcf pathway results in an accelerated degradation of the wild-type beta-catenin, suggesting that the negative feedback loop regulation may control the beta-catenin/Tcf pathway. This signaling also upregulated NF-kappaB transactivation without affecting the activity of IkappaB kinase, thereby establishing that the maintenance of the betaTrCP level is important for coordination between beta-catenin/Tcf and NF-kappaB signaling.

Development and initial characterization of several new inbred strains of SENCAR mice for studies of multistage skin carcinogenesis.

The development and initial characterization of five new inbred strains of SENCAR mice are described in this paper. Ten randomly selected pairs of outbred SENCAR mice were mated and offspring from each separately maintained parental line were sib mated at each successive generation to result in inbred strains. Due to poor reproductive performance only five of the original 10 lines were bred to homogeneity. Initial characterization of the five remaining lines (referred to as SL2/sprd, SL5/sprd, SL7/sprd, SL8/sprd and SLl0/sprd) at F12 for their responsiveness to a two-stage carcinogenesis protocol (10 nmol 7,12-dimethylbenz[a]anthracene and 0.25 microg 12-O-tetradecanoylphorbol-13 acetate) revealed three groups of responders in terms of the number of papillomas per mouse: SL2/sprd and SL8/sprd > SL7/sprd and SL10/sprd >> SL5/sprd. The papilloma responses in SL2/sprd and SL8/sprd were very similar to SENCAR B/Pt compared at the same doses. Papillomas induced on SL2/sprd had the highest propensity to progress to squamous cell carcinomas, similar to that observed in outbred SENCAR and SENCAR B/Pt mice. More detailed comparison of the responsiveness of SL2/sprd and SL5/sprd at Fl5 showed that these two inbred strains differed in their sensitivity to TPA-induced epidermal hyperplasia and that the dose of TPA required to produce a tumor response in SL5/sprd in comparison with that in SL2/sprd was 4-20 times higher. Overall, the availability of the different inbred SENCAR strains will greatly aid mechanistic studies of multistage skin carcinogenesis as well as studies to understand the underlying genetic basis of resistance to tumor promotion and progression in this model system.

Inhibition of azoxymethane-induced rat colon carcinogenesis by potassium hydrogen D-glucarate.

While calcium D-glucarate was shown to inhibit chemical carcinogenesis in various animal models, the effect of potassium hydrogen D-glucarate has not been extensively investigated. In the present study, potassium hydrogen D-glucarate markedly inhibited azoxymethane (AOM)-induced colon carcinogenesis in male F344 rats. Potassium hydrogen D-glucarate (PHG) or potassium hydrogen carbonate (PHC) were administered to rats in a diet (140 mmol/kg). Continual post-initiation treatment with potassium hydrogen D-glucarate reduced both tumor incidence and multiplicity at sacrifice by ca. 60%, while PHC had no effect. amelioration of overexpression of the betaG gene in rat colon carcinomas was observed using RT-PCR and Northern blot analysis. We hypothesize that previously demonstrated conversion of PHG to D-glucaro-1,4-lactone, a potent inhibitor of beta-glucuronidase (betaG), may be responsible for this effect. The mechanism of PHG inhibition of colon carcinogenesis may also involve suppression of cell proliferation and possibly alterations in cholesterol synthesis or cholesterol metabolism to bile acids. In conclusion, PHG possesses excellent potential as a natural, apparently non-toxic inhibitor to prevent colon cancer.

Increased expression of p50-NF-kappaB and constitutive activation of NF-kappaB transcription factors during mouse skin carcinogenesis.

To elucidate the possible role of NF-kappaB in mouse skin carcinogenesis we studied the expression of p50 (NF-kappaB1), p52 (NF-kappaB2), p65 (RelA) and IkappaB-alpha inhibitor as well as kappaB-binding activity in adult SENCAR mouse skin, skin papillomas, and squamous cell carcinomas (SCC) generated by a two-stage carcinogenesis protocol. We found that in normal epidermis all of the above proteins were mostly expressed in the cytoplasm of basal cells. Western blot analysis revealed a dramatic increase of p50 and p52 expression in mouse skin tumors starting from the middle stage of promotion. We also found that the level of IkappaB-alpha protein in many late papillomas and SCC was lower than in normal epidermis. Results of EMSA showed an increase in kappaB-binding activity in mouse skin tumors and suggested that p50 is the major component of constitutive kappaB-binding complexes in normal epidermis and in tumors. It has been shown that nuclear IkappaB protein Bcl-3 is able to increase p50/p50 homodimer binding to the different kappaB sites in mouse thymocytes. Our finding on Bcl-3 overexpression in late papillomas and SCC could explain the selective increase of p50-related kappaB-binding in mouse skin tumors. Thus, our results strongly suggest the important role of p50 in skin carcinogenesis.

Tumor promoter benzoyl peroxide induces sulfhydryl oxidation in protein kinase C: its reversibility is related to the cellular resistance to peroxide-induced cytotoxicity.

Since tumor promoter benzoyl peroxide (BPO) mimics phorbol esters in some aspects, its effects on protein kinase C (PKC) were previously studied. However, in those studies due to the presence of thiol agents in the PKC preparations, the sensitive reaction of BPO with redox-active cysteine residues in PKC was not observed. In this study, by excluding thiol agents present in the purified PKC preparation, low concentrations of BPO modified PKC, resulting in the loss of both kinase activity and phorbol ester binding (IC50 = 0. 2 to 0.5 microM). This modification, which was not dependent on transition metals, was totally blocked by a variety of thiol agents including GSH, which directly reacted with BPO. Substoichiometric amounts of BPO (0.4 mol/mol of PKC) oxidized two sulfhydryls in PKC and inactivated the enzyme which was readily reversed by dithiothreitol. The regulatory domain having zinc thiolate structures supporting the membrane-inserting region provided the specificity for PKC reaction with BPO, which partitioned into the membrane. Unlike H2O2, BPO did not induce the generation of the Ca2+/lipid-independent activated form of PKC. Other redox-sensitive enzymes such as protein kinase A, phosphorylase kinase, and protein phosphatase 2A required nearly 25- to 100-fold higher concentrations of BPO for inactivation. BPO also inactivated PKC in a variety of cell types. In the JB6 (30 P-) nonpromotable cell line and other normal cell lines, where BPO was more cytotoxic, it readily inactivated PKC due to a slow reversibility of this inactivation by the cell. However, in the JB6 (41 P+) promotable cell line, C3H10T1/2 and B16 melanoma cells, where BPO was less cytotoxic, it did not readily inactivate PKC due to a rapid reversibility of this inactivation by an endogenous mechanism. Nevertheless, BPO inactivated PKC at an equal rate in the homogenates prepared from all these cell types. Inclusion of NADPH reversed this inactivation in the homogenates to a different extent, presumably due to a difference in distribution of a protein disulfide reductase, which reverses this oxidative modification. BPO-induced modification of PKC occurred independent of the cellular status of GSH. However, externally added GSH and cell-impermeable thiol agents prevented the BPO-induced modification of PKC. Since BPO readily partitions into membranes, its reaction with redox-cycling thiols of membrane proteins such as PKC may trigger epigenetic events to prevent cytotoxicity, but favor tumor promotion.

Mechanism-based cancer prevention approaches: targets, examples, and the use of transgenic mice.

Humans are exposed to a wide variety of carcinogenic insults, including endogenous and man-made chemicals, radiation, physical agents, and viruses. The ultimate goal of carcinogenesis research is to elucidate the processes involved in the induction of human cancer so that interventions may be developed to prevent the disease, either in the general population or in susceptible subpopulations. Progress to date in the carcinogenesis field, particularly regarding the mechanisms of chemically induced cancer, has revealed several points along the carcinogenesis pathway that may be amenable to mechanism-based prevention strategies. The purpose of this review is to examine the basic mechanisms and stages of chemical carcinogenesis, with an emphasis on ways in which preventive interventions can modify those processes. Possible ways of interfering with tumor initiation events include the following: i) modifying carcinogen activation by inhibiting enzymes responsible for that activation or by direct scavenging of DNA-reactive electrophiles and free radicals; ii) enhancing carcinogen detoxification processes by altering the activity of the detoxifying enzymes; and iii) modulating certain DNA repair processes. Possible ways of blocking the processes involved in the promotion and progression stages of carcinogenesis include the following: i) scavenging of reactive oxygen species; ii) altering the expression of genes involved in cell signaling, particularly those regulating cell proliferation, apoptosis, and differentiation; and iii) decreasing inflammation. In addition, the utility for mechanism-based cancer prevention research of new animal models that are based on the overexpression or inactivation of specific cancer-related genes is examined.

Short-term biomarkers of tumor promotion in mouse skin treated with petroleum middle distillates.

Topical application of certain petroleum middle distillates (PMD) to mice produces skin tumors after long latency, and initiation/promotion protocols indicate that this effect is associated with their tumor promoting activity. Since induction of sustained, potentiated epidermal hyperplasia is predictive of promoting activity, five compositionally distinct PMD [hydrodesulfurized kerosene (API 81-07); hydrodesulfurized PMD (API 81-10); odorless light petroleum hydrocarbons; severely hydrotreated light vacuum distillate (LVD); and lightly refined paraffinic oil (LRPO)] were assessed for their effects on epidermal hyperplasia. PMD were administered (2 x/week for 2 weeks) to skin of CD-1 mice. Four quantitative biomarkers of epidermal hyperplasia were evaluated: epidermal thickness, number of nucleated epidermal cells per unit length of basement membrane, labeling (BrdUrd) index of epidermal cells, and induction of epidermal ornithine decarboxylase (ODC) activity. As positive controls, 12-O-tetradecanoylphorbol-13-acetate (TPA) and n-dodecane were utilized. PMD-induced skin irritation was evaluated visually and/or histopathologically. All five PMD produced dose-dependent, skin irritation and epidermal hyperplasia. On a weight basis the magnitude of the maximal PMD-induced effects was similar to that produced by n-dodecane, but > 1000-fold less than that produced by TPA. Epidermal hyperplasia and subacute skin irritancy produced by the five PMD were similar. Of the four short-term markers of tumor promotion assessed, labeling index and epidermal ODC activity were predictive of the relative promoting activities of those PMD for which tumorigenicity bioassay data are available, i.e., API 81-07 > API 81-10 > LRPO. An apparent discrepancy to the predictability of epidermal ODC activity occurred with LRPO:toluene [1:1 (v/v)]. This mixture is nontumorigenic, yet significantly induced epidermal ODC activity. This mixture, however, produced severe epidermal toxicity that precluded any meaningful analysis of short-term biomarkers in relationship to biological activity.

Comparison of the skin tumor-promoting potential of different organic peroxides in SENCAR mice.

The skin tumor-promoting activities of three organic peroxides were evaluated and compared to the activity of benzoyl peroxide, a well-characterized tumor promoter. Two of the compounds (di-t-butyl peroxide and dicumyl peroxide) were dialkyl peroxides and the other (di-m-chlorobenzoyl peroxide) was a diacyl peroxide. These compounds were selected based on a previous study in which we evaluated their capacity to induce epidermal hyperplasia, ornithine decarboxylase activity, and dark basal keratinocytes, which have been reliable short-term markers of tumor promotion. Dicumyl peroxide was a weak tumor promoter despite its high activity in inducing hyperplasia. Like benzoyl peroxide, di-m-chlorobenzoyl peroxide generally had intermediate activity as an inducer of short-term markers of tumor promotion and was a moderately effective tumor promoter. However, compared to benzoyl peroxide, di-m-chlorobenzoyl peroxide was more toxic to the skin, which may have limited its tumor-promoting activity. The final compound, di-t-butyl peroxide, which was essentially inactive in short-term assays, was also totally inactive in promoting papillomas or carcinomas in initiated skin. Tumor-promoting efficacy generally showed an inverse association with thermal stability for the compounds tested, suggesting that the rate of formation of free radicals is a key factor contributing to tumor promotion by organic peroxides. However, a number of other factors can potentially affect the activity of different organic peroxides as tumor promoters. Each compound evaluated had a different spectrum of activities, and these compounds should be useful for studying mechanisms of organic peroxide-induced tumor promotion.

Analysis of two inbred strains of mice derived from the SENCAR stock with different susceptibility to skin tumor progression.

The SENCAR stock of mice has proved to be a useful model in dissecting out the multistage nature as well as the critical mechanisms involved in skin tumorigenesis. This outbred stock was selectively bred to be susceptible to initiation with 7,12-dimethylbenz[a]anthracene (DMBA) and promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA). In order to obtain mice more suitable for genetic analyses of tumor susceptibility and tissue transplantation studies, several inbred lines of mice were derived from the SENCAR stock. One of these lines, the SSIN mice, has a higher susceptibility to tumor promotion compared to the SENCAR stock but is very resistant to tumor progression. On the other hand, the SENCAR B/Pt mice, derived also from the outbred stock, not only have a tumor promotion susceptibility almost identical to the SSIN mice, but they also have a high susceptibility to tumor progression. In order to understand the nature of the phenotypic differences between these two inbred lines we have characterized them using several parameters and markers that are associated with the progression of papillomas to squamous cell carcinoma (SCC). In this sense we analysed the tumor multiplicity and SCC incidence, and the expression of markers of progression and cell cycle related proteins in papillomas derived from both strains. Our results showed that while both strains have a similar papilloma multiplicity and incidence the SENCAR B/Pt mice have 67% incidence of SCC, compared to 0% in the SSIN. SENCAR B/Pt papillomas at 30 weeks of promotion have a higher and aberrant expression of K13, and loss of connexin 26. TGF-beta1 was found to be over-expressed in the suprabasal and superficial cells in the SENCAR B/Pt papillomas, while it was only expressed in the superficial cell layer in those derived from SSIN. The SENCAR B/Pt papillomas also showed an enlarged proliferative compartment with overexpression of cyclin D1 and PCNA as seen by immunohistochemistry and Western blot.

Resistance of transformed mouse keratinocytes to growth inhibition by glucocorticoids.

Glucocorticoid hormones are strong inhibitors of normal keratinocyte proliferation, but established mouse skin papillomas and carcinomas become resistant to these hormones. The biological effect of glucocorticoids is mediated through a highly specific glucocorticoid receptor (GR). To study the possible mechanisms of glucocorticoid resistance of transformed mouse keratinocytes, we evaluated GR expression and function in non-tumorigenic (3PC), papilloma-producing (MT1/2 and P1/17), and squamous cell carcinoma-producing (Ca3/7 and Ca8/29) keratinocyte cell lines and analyzed the DNA sequence of GR in glucocorticoid-sensitive and glucocorticoid-resistant keratinocytes. All transformed keratinocyte cell lines studied appeared to be completely resistant to the growth inhibition by the glucocorticoid fluocinolone acetonide (FA), whereas the untransformed cell line 3PC was very sensitive to FA. Despite the glucocorticoid resistance, all the tumorigenic keratinocyte cell lines expressed high levels of GR mRNA and protein. Southern blot analysis and direct sequencing of the DNA-binding domain of the GR gene revealed no significant changes in GR gene structure in transformed keratinocytes. To test the functional capability of GR, we compared the effect of FA on the expression of glucocorticoid-responsive genes. FA strongly induced metallothionein 1 expression in 3PC cells, slightly induced metallothionein 1 expression in P1/17 and Ca3/7 cells, and did not affect its expression in MT1/2 and Ca8/29 cells. These data suggest that resistance to the growth inhibition of glucocorticoids is an important feature of tumorigenic keratinocyte cell lines. It is likely that this hormone-resistant phenotype is a result of alteration of GR function but not of GR expression or gene structure.

Changes in protein expression during multistage mouse skin carcinogenesis.

To directly compare the expression patterns of different proteins known to be altered during mouse skin carcinogenesis, serial sections of normal and hyperplastic skin and tumors from various stages of 7,12-dimethylbenz[a]anthracene-initiated, 12-O-tetradecanoylphorbol-13-acetate-promoted female SENCAR mice were examined by immunohistochemistry. In untreated, normal mouse skin, keratin 1 (K1) and transforming growth factor-beta1 (TGFbeta1) were strongly expressed in the suprabasal layers, whereas integrin alpha6beta4 was expressed only in basal cells and only moderate staining for transforming growth factor-alpha (TGFalpha) was seen. In hyperplastic skin, TGFalpha expression became stronger, whereas expression of another epidermal growth factor (EGF) receptor ligand, heparin-binding EGF-like growth factor (HB-EGF), was strongly induced in all epidermal layers from no expression in normal skin. Likewise, the gap-junctional protein connexin 26 (Cx26) became highly expressed in the differentiated granular layers of hyperplastic skin relative to undetectable expression in normal skin. Expression of cyclin D1 in the proliferative cell compartment was seen in all benign and malignant tumors but not in hyperplastic skin. Beginning with very early papillomas (after 10 wk of promotion), expression of alpha6beta4 in suprabasal cells and small, focal staining for keratin 13 (K13) were seen in some tumors. Later (after 20-30 wk), focal areas of gamma-glutamyl transpeptidase (GGT) activity appeared in a few papillomas, whereas TGFbeta1 expression began to decrease. Cx26 and TGFalpha staining became patchier in some late-stage papillomas (30-40 wk), whereas suprabasal alpha6beta4, K13, and GGT expression progressively increased and K1 expression decreased. Finally, in squamous cell carcinomas (SCCs), there was an almost complete loss of K1 and a further decline in TGFalpha, HB-EGF, TGFbeta1, and Cx26 expression. On the other hand, almost all SCCs showed suprabasal staining for alpha6beta4 and widespread cyclin D1 and K13 expression, whereas only about half showed positive focal staining for GGT activity.

Altered glucocorticoid receptor expression and function during mouse skin carcinogenesis.

Glucocorticoids are the most potent inhibitors of tumor promotion in mouse skin, when applied with a promoting agent at the early stages of promotion. However, established skin papillomas become resistant to growth inhibition by glucocorticoids. Glucocorticoid control of cellular functions is mediated by the glucocorticoid receptor (GR), a well-known transcription factor. Here we present data on GR expression and function in mouse papillomas and squamous cell carcinomas. Tumors were produced in SENCAR mice by a 7,12-dimethylbenz[a]anthracene and 12-O-tetradecanoylphorbol-13-acetate two-stage protocol. In early papillomas (after 15-20 wk of promotion), northern blotting revealed a decrease in the GR mRNA level that was confirmed by a binding assay. However, in late papillomas (after 30-40 wk of promotion), and especially in squamous cell carcinomas, the level of GR in both assays was similar to or higher than the GR level in normal epidermis. To test the functional capability of GR in tumors, we compared the effect of the synthetic glucocorticoid fluocinolone acetonide (FA) on keratinocyte proliferation and on expression of glucocorticoid-responsive genes in normal epidermis, hyperplastic skin surrounding tumors, and mouse skin papillomas. FA strongly inhibited DNA synthesis in keratinocytes in normal skin and tumor-surrounding skin but had no effect on DNA synthesis in papillomas. In addition, FA strongly induced metallothionein 1 expression and inhibited connexin 26 expression in skin but did not affect expression of these genes in tumors. These data suggest that alteration of both the expression and function of GR may be an important mechanism of tumor promotion in skin.

Induction of mammary cancer and lymphoma by multiple, low oral doses of 7,12-dimethylbenz[a]anthracene in SENCAR mice.

Existing models of mouse mammary carcinogenesis induced by the model polycyclic aromatic hydrocarbon 7,12-dimethylbenz[a]anthracene (DMBA) typically use a small number of bolus doses applied intragastrically. In contrast to this, typical human exposures to carcinogens are thought to be at lower doses and to occur with chronic or sporadic timing. When the classical dosage (1 mg DMBA given once a week for 6 weeks) was split into five daily doses of 200 microg given intragastrically to female SENCAR mice each week for 6 weeks, toxicity was high and the major tumor type seen was lymphoma. Lowering the dose to 60 microg/day gave less toxicity, a 75% incidence of lymphoma and a 30% incidence of mammary carcinoma. However, 20 microg DMBA given five times per week for 6 weeks resulted in a 65-70% incidence of mammary carcinoma within approximately 50 weeks. This represents a 50-fold lower daily dosage of DMBA than that used in the classical model. DNA was prepared from 10 mammary adenocarcinomas and 10 lymphomas and exons 1 and 2 of the H-ras1, K-ras and N-ras genes were sequenced using PCR techniques. Mutations altering codons 12 or 61 of one of the ras family genes were found in 4/10 mammary carcinomas and 5/10 lymphomas. Three mammary tumors exhibited codon 61 mutations, one in each of the genes studied, and a fourth tumor contained a codon 12 mutation in the K-ras gene. Among the lymphomas, two mutations in codon 12 of K-ras, one mutation in codon 61 of K-ras and two mutations in codon 61 of N-ras were also found. Each of the mutations could be interpreted as a G-->T or A-->T transversion. It is suggested that the high incidence of lymphoma at the higher, repetitive doses may be related to immunotoxicity. These low dose models of lymphomagenesis and mammary carcinogenesis should prove useful for tests of chemopreventive agents that target the initiation phase of carcinogenesis.