Erika L. Abel

Multi-stage chemical carcinogenesis in mouse skin: Fundamentals and applications

For more than 60 years, the chemical induction of tumors in mouse skin has been used to study mechanisms of epithelial carcinogenesis and evaluate modifying factors. In the traditional two-stage skin carcinogenesis model, the initiation phase is accomplished by the application of a sub-carcinogenic dose of a carcinogen. Subsequently, tumor development is elicited by repeated treatment with a tumor-promoting agent. The initiation protocol can be completed within 1–3 h depending on the number of mice used; whereas the promotion phase requires twice weekly treatments (1–2 h) and once weekly tumor palpation (1–2 h) for the duration of the study. Using the protocol described here, a highly reproducible papilloma burden is expected within 10–20 weeks with progression of a portion of the tumors to squamous cell carcinomas within 20–50 weeks. In contrast to complete skin carcinogenesis, the two-stage model allows for greater yield of premalignant lesions, as well as separation of the initiation and promotion phases.

Differential gene expression in epidermis of mice sensitive and resistant to phorbol ester skin tumor promotion

Previous data from two‐stage carcinogenesis studies in mouse skin demonstrated that genetic control of susceptibility to skin tumor promotion by the phorbol ester, 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), in crosses between susceptible DBA/2J and resistant C57BL/6J mice is a multigenic trait. Utilizing a cDNA microarray approach, we compared global gene expression profiles in the epidermis of these two mouse strains treated with TPA or vehicle (acetone). Gene expression in the epidermis was analyzed after the treatment to identify global effects of TPA, as well as potential candidate genes that modify susceptibility to skin tumor promotion. DBA/2J and C57BL/6J mice were treated topically four times with 3.4 nmol TPA or acetone over a 2‐wk period, and RNA was extracted from epidermis 6 h after the final treatment. Labeled cDNA generated from each group was hybridized to commercial cDNA microarrays (Agilent) containing more than 8000 targets. More than 450 genes were significantly influenced, directly or indirectly, by TPA treatment in the epidermis of either strain. Notably, 44 genes exhibited differential expression between the tumor promotion sensitive and resistant mouse strains. Several genes that were differentially expressed in DBA/2J versus C57BL/6J epidermis after TPA treatment were located in chromosomal regions linked to TPA promotion susceptibility. Three genes, Gsta4, Nmes1 (MGC58382), and Serpinb2, located within promotion susceptibility loci Psl1 (chr 9), Psl2 (chr 2), and Psl3 (chr 1), respectively, were identified in this analysis as potential candidates for modifiers of susceptibility to skin tumor promotion by TPA.

Evidence That Gsta4 Modifies Susceptibility to Skin Tumor Development in Mice and Humans

BACKGROUND The incidence of nonmelanoma skin cancer (NMSC) is equivalent to that of all other cancers combined. Previously, we mapped the 12-O-tetradecanoylphorbol-13-acetate (TPA) skin tumor promotion susceptibility locus, Psl1, to distal chromosome 9 in crosses of sensitive DBA/2 mice with relatively resistant C57BL/6 mice. Here, we used the mouse two-stage skin carcinogenesis model to identify the gene(s) responsible for the effects of Psl1. METHODS Interval-specific congenic mouse strains (n ≥ 59 mice per strain) were used to more precisely map the Psl1 locus. Having identified glutathione S-transferase α4 (Gsta4) as a candidate tumor promotion susceptibility gene that mapped within the delimited region, we analyzed Gsta4-deficient mice (n = 62) for susceptibility to skin tumor promotion by TPA. We used quantitative polymerase chain reaction, western blotting, and immunohistochemistry to verify induction of Gsta4 in mouse epidermis following TPA treatment and biochemical assays to associate Gsta4 activity with tumor promotion susceptibility. In addition, single-nucleotide polymorphisms (SNPs) in GSTA4 were analyzed in a case-control study of 414 NMSC patients and 450 control subjects to examine their association with human NMSC. Statistical analyses of tumor studies in mice were one-sided, whereas all other statistical analyses were two-sided. RESULTS Analyses of congenic mice indicated that at least two loci, Psl1.1 and Psl1.2, map to distal chromosome 9 and confer susceptibility to skin tumor promotion by TPA. Gsta4 maps to Psl1.2 and was highly induced (mRNA and protein) in the epidermis of resistant C57BL/6 mice compared with that of sensitive DBA/2 mice following treatment with TPA. Gsta4 activity levels were also higher in the epidermis of C57BL/6 mice following treatment with TPA. Gsta4-deficient mice (C57BL/6.Gsta4(-/-) mice) were more sensitive to TPA skin tumor promotion (0.8 tumors per mouse vs 0.4 tumors per mouse in wild-type controls; difference = 0.4 tumors per mouse; 95% confidence interval = 0.1 to 0.7, P = .007). Furthermore, inheritance of polymorphisms in GSTA4 was associated with risk of human NMSC. Three SNPs were found to be independent predictors of NMSC risk. Two of these were associated with increased risk of NMSC (odds ratios [ORs] = 1.60 to 3.42), while the third was associated with decreased risk of NMSC (OR = 0.63). In addition, a fourth SNP was associated with decreased risk of basal cell carcinoma only (OR = 0.44). CONCLUSIONS Gsta4/GSTA4 is a novel susceptibility gene for NMSC that affects risk in both mice and humans.

Protection against 2-chloroethyl ethyl sulfide (CEES) — induced cytotoxicity in human keratinocytes by an inducer of the glutathione detoxification pathway

Sulfur mustard (SM or mustard gas) was first used as a chemical warfare agent almost 100years ago. Due to its toxic effects on the eyes, lungs, and skin, and the relative ease with which it may be synthesized, mustard gas remains a potential chemical threat to the present day. SM exposed skin develops fluid filled bullae resulting from potent cytotoxicity of cells lining the basement membrane of the epidermis. Currently, there are no antidotes for SM exposure; therefore, chemopreventive measures for first responders following an SM attack are needed. Glutathione (GSH) is known to have a protective effect against SM toxicity, and detoxification of SM is believed to occur, in part, via GSH conjugation. Therefore, we screened 6 potential chemopreventive agents for ability to induce GSH synthesis and protect cultured human keratinocytes against the SM analog, 2-chloroethyl ethyl sulfide (CEES). Using NCTC2544 human keratinocytes, we found that both sulforaphane and methyl-2-cyano-3,12-dioxooleana-1,9-dien-28-oate (CDDO-Me) stimulated nuclear localization of Nrf2 and induced expression of the GSH synthesis gene, GCLM. Additionally, we found that treatment with CDDO-Me elevated reduced GSH content of NCTC2544 cells and preserved their viability by ~3-fold following exposure to CEES. Our data also suggested that CDDO-Me may act additively with 2,6-dithiopurine (DTP), a nucleophilic scavenging agent, to increase the viability of keratinocytes exposed to CEES. These results suggest that CDDO-Me is a promising chemopreventive agent for SM toxicity in the skin.

Upregulation of the EP1 receptor for prostaglandin E2 promotes skin tumor progression

Prostaglandin E2 (PGE2) has been shown to promote the development of murine skin tumors. EP1 is 1 of the 4 PGE2 G‐protein‐coupled membrane receptors expressed by murine keratinocytes. EP1 mRNA levels were increased ∼2‐fold after topical treatment with 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) or exposure to ultraviolet (UV) light, as well as increased ∼3‐ to 12‐fold in tumors induced by 7,12‐dimethyl‐benz[a]anthracene (DMBA) initiation/TPA promotion or by UV exposure. To determine the effect of EP1 levels on tumor development, we generated BK5.EP1 transgenic mice that overexpress EP1 in the basal layer of the epidermis. Skins of these mice were histologically indistinguishable from wild type (WT) mice and had similar levels of proliferation after TPA treatment. Using a DMBA/TPA carcinogenesis protocol, BK5.EP1 mice had a reduced tumor multiplicity compared to WT mice, likely due to the observed down‐regulation of protein kinase C (PKC). However, the BK5.EP1 mice had an ∼8‐fold higher papilloma to carcinoma conversion rate. When DMBA/anthralin was used, BK5.EP1 mice produced more tumors than WT mice, as well as a ninefold increase in carcinomas, indicating that the tumor response is dependent on the type of tumor promoter agent used. Additionally, although almost undetectable in WT mice, cyclooxygenase‐2 (COX‐2) was expressed in the untreated epidermis of BK5.EP1 mice. While TPA highly induced COX‐2 in WT mice, COX‐2 expression in the BK5.EP1 mice did not change after TPA treatment; PGE2 levels were likewise affected. These data indicate that EP1 is more important in tumor progression than in tumor promotion and that it indirectly regulates COX‐2 expression.

A high-throughput 1,536-well luminescence assay for glutathione S-transferase activity.

Glutathione S-transferases (GSTs) constitute a family of detoxification enzymes that catalyze the conjugation of glutathione with a variety of hydrophobic compounds, including drugs and their metabolites, to yield water-soluble derivatives that are excreted in urine or bile. Profiling the effect of small molecules on GST activity is an important component in the characterization of drug candidates and compound libraries. Additionally, specific GST isozymes have been implicated in drug resistance, especially in cancer, and thus represent potential targets for intervention. To date, there are no sensitive miniaturized high-throughput assays available for GST activity detection. A series of GST substrates containing a masked luciferin moiety have been described recently, offering the potential for configuring a sensitive screening assay via coupled luciferase reaction and standard luminescence detection. We report on the optimization and miniaturization of this homogeneous method to 1,536-well format using GSTs from 3 different species: mouse isozyme A4-4, human isozymes A1-1, M1-1, and P1-1, and the major GST from the parasitic worm Schistosoma japonicum.

Sulforaphane induces phase II detoxication enzymes in mouse skin and prevents mutagenesis induced by a mustard gas analog

Mustard gas, used in chemical warfare since 1917, is a mutagenic and carcinogenic agent that produces severe dermal lesions for which there are no effective therapeutics; it is currently seen as a potential terrorist threat to civilian populations. Sulforaphane, found in cruciferous vegetables, is known to induce enzymes that detoxify compounds such as the sulfur mustards that react through electrophilic intermediates. Here, we observe that a single topical treatment with sulforaphane induces mouse epidermal levels of the regulatory subunit of glutamate-cysteine ligase, the rate-limiting enzyme in glutathione biosynthesis, and also increases epidermal levels of reduced glutathione. Furthermore, a glutathione S-transferase, GSTA4, is also induced in mouse skin by sulforaphane. In an in vivo model in which mice are given a single mutagenic application of the sulfur mustard analog 2-(chloroethyl) ethyl sulfide (CEES), we now show that therapeutic treatment with sulforaphane abolishes the CEES-induced increase in mutation frequency in the skin, measured four days after exposure. Sulforaphane, a natural product currently in clinical trials, shows promise as an effective therapeutic against mustard gas.

Proteomic and pathway analyses reveal a network of inflammatory genes associated with differences in skin tumor promotion susceptibility in DBA/2 and C57BL/6 mice

Genetic susceptibility to two-stage skin carcinogenesis is known to vary significantly among different stocks and strains of mice. In an effort to identify specific protein changes or altered signaling pathways associated with skin tumor promotion susceptibility, a proteomic approach was used to examine and identify proteins that were differentially expressed in epidermis between promotion-sensitive DBA/2 and promotion-resistant C57BL/6 mice following treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). We identified 19 differentially expressed proteins of which 5 were the calcium-binding proteins annexin A1, parvalbumin α, S100A8, S100A9, and S100A11. Further analyses revealed that S100A8 and S100A9 protein levels were also similarly differentially upregulated in epidermis of DBA/2 versus C57BL/6 mice following topical treatment with two other skin tumor promoters, okadaic acid and chrysarobin. Pathway analysis of all 19 identified proteins from the present study suggested that these proteins were components of several networks that included inflammation-associated proteins known to be involved in skin tumor promotion (e.g. TNF-α, NFκB). Follow-up studies revealed that Tnf, Nfkb1, Il22, Il1b, Cxcl1, Cxcl2 and Cxcl5 mRNAs were highly expressed in epidermis of DBA/2 compared with C57BL/6 mice at 24h following treatment with TPA. Furthermore, NFκB (p65) was also highly activated at the same time point (as measured by phosphorylation at ser276) in epidermis of DBA/2 mice compared with C57BL/6 mice. Taken together, the present data suggest that differential expression of genes involved in inflammatory pathways in epidermis may play a key role in genetic differences in susceptibility to skin tumor promotion in DBA/2 and C57BL/6 mice.

2,6-Dithiopurine, a Nucleophilic Scavenger, Protects Against Mutagenesis in Mouse Skin Treated in vivo with 2-(chloroethyl) ethyl sulfide, a Mustard Gas Analog

Sulfur mustard [bis(2-chloroethyl)sulfide, SM] is a well-known DNA-damaging agent that has been used in chemical warfare since World War I, and is a weapon that could potentially be used in a terrorist attack on a civilian population. Dermal exposure to high concentrations of SM produces severe, long-lasting burns. Topical exposure to high concentrations of 2-(chloroethyl) ethyl sulfide (CEES), a monofunctional analog of SM, also produces severe skin lesions in mice. Utilizing a genetically engineered mouse strain, Big Blue, that allows measurement of mutation frequencies in mouse tissues, we now show that topical treatment with much lower concentrations of CEES induces significant dose- and time-dependent increases in mutation frequency in mouse skin; the mutagenic exposures produce minimal toxicity as determined by standard histopathology and immunohistochemical analysis for cytokeratin 6 and the DNA-damage induced phosphorylation of histone H2AX (γ-H2AX). We attempted to develop a therapeutic that would inhibit the CEES-induced increase in mutation frequency in the skin. We observe that multi-dose, topical treatment with 2,6-dithiopurine (DTP), a known chemical scavenger of CEES, beginning 1h post-exposure to CEES, completely abolishes the CEES-induced increase in mutation frequency. These findings suggest the possibility that DTP, previously shown to be non-toxic in mice, may be useful as a therapeutic agent in accidental or malicious human exposures to SM.

The EP1 receptor for prostaglandin E2 promotes the development and progression of malignant murine skin tumors

High levels of prostaglandin E2 (PGE2) synthesis resulting from the up‐regulation of cyclooxygenase (COX)‐2 has been shown to be critical for the development of non‐melanoma skin tumors. This effect of PGE2 is likely mediated by one or more of its 4 G‐protein coupled membrane receptors, EP1‐4. A previous study showed that BK5.EP1 transgenic mice produced more carcinomas than wild type (WT) mice using initiation/promotion protocols, although the tumor response was dependent on the type of tumor promoter used. In this study, a single topical application of either 7,12‐dimethylbenz[a]anthracene (DMBA) or benzo[a]pyrene (B[a]P), alone, was found to elicit squamous cell carcinomas (SCCs) in the BK5.EP1 transgenic mice, but not in WT mice. While the epidermis of both WT and transgenic mice was hyperplastic several days after DMBA, this effect regressed in the WT mice while proliferation continued in the transgenic mice. Several parameters associated with carcinogen initiation were measured and were found to be similar between genotypes, including CYP1B1 and aromatase expression, B[a]P adduct formation, Ras activity, and keratinocyte stem cell numbers. However, EP1 transgene expression elevated COX‐2 levels in the epidermis and SCC could be completely prevented in DMBA‐treated BK5.EP1 mice either by feeding the selective COX‐2 inhibitor celecoxib in their diet or by crossing them onto a COX‐2 null background. These data suggest that the tumor promoting/progressing effects of EP1 require the PGE2 synthesized by COX‐2.