Chemical Carcinogenesis In Mouse Skin Research Articles

Loss of Snail2 favors skin tumor progression by promoting the recruitment of myeloid progenitors

Snail2 is a zinc finger transcription factor involved in driving epithelial to mesenchymal transitions. Snail2 null mice are viable, but display defects in melanogenesis, gametogenesis and hematopoiesis, and are markedly radiosensitive. Here, using mouse genetics, we have studied the contributions of Snail2 to epidermal homeostasis and skin carcinogenesis. Snail2 (-/-) mice presented a defective epidermal terminal differentiation and, unexpectedly, an increase in number, size and malignancy of tumor lesions when subjected to the two-stage mouse skin chemical carcinogenesis protocol, compared with controls. Additionally, tumor lesions from Snail2 (-/-) mice presented a high inflammatory component with an elevated percentage of myeloid precursors in tumor lesions that was further increased in the presence of the anti-inflammatory agent dexamethasone. In vitro studies in Snail2 null keratinocytes showed that loss of Snail2 leads to a decrease in proliferation indicating a non-cell autonomous role for Snail2 in the skin carcinogenic response observed in vivo. Bone marrow (BM) cross-reconstitution assays between Snail2 wild-type and null mice showed that Snail2 absence in the hematopoietic system fully reproduces the tumor behavior of the Snail2 null mice and triggers the accumulation of myeloid precursors in the BM, blood and tumor lesions. These results indicate a new role for Snail2 in preventing myeloid precursors recruitment impairing skin chemical carcinogenesis progression.

Keratinocyte p38δ loss inhibits Ras-induced tumor formation, while systemic p38δ loss enhances skin inflammation in the early phase of chemical carcinogenesis in mouse skin.

p38δ expression and/or activity are increased in human cutaneous malignancies, including invasive squamous cell carcinoma (SCC) and head and neck SCC, but the role of p38δ in cutaneous carcinogenesis has not been well-defined. We have reported that mice with germline loss of p38δ exhibited a reduced susceptibility to skin tumor development compared with wild-type mice in the two-stage 7,12-dimethylbenz(a)anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) chemical skin carcinogenesis model. Here, we report that p38δ gene ablation inhibited the growth of tumors generated from v-ras(Ha) -transformed keratinocytes in skin orthografts to nude mice, indicating that keratinocyte-intrinsic p38δ is required for Ras-induced tumorigenesis. Gene expression profiling of v-ras(Ha) -transformed p38δ-null keratinocytes revealed transcriptional changes associated with cellular responses linked to tumor suppression, such as reduced proliferation and increased differentiation, cell adhesion, and cell communications. Notably, a short-term DMBA/TPA challenge, modeling the initial stages of chemical skin carcinogenesis treatment, elicited an enhanced inflammation in p38δ-null skin compared with skin of wild-type mice, as assessed by measuring the expression of pro-inflammatory cytokines, including IL-1β, IL-6, IL-17, and TNFα. Additionally, p38δ-null skin and p38δ-null keratinocytes exhibited increased p38α activation and signaling in response to acute inflammatory challenges, suggesting a role for p38α in stimulating the elevated inflammatory response in p38δ-null skin during the initial phases of the DMBA/TPA treatment compared with similarly treated p38δ(+/+) skin. Altogether, our results indicate that p38δ signaling regulates skin carcinogenesis not only by keratinocyte cell-autonomous mechanisms, but also by influencing the interaction between between the epithelial compartment of the developing skin tumor and its stromal microenvironment.

Inhibitory Effects of Opuntia humifusa on 7, 12-Dimethyl-benz[a]anthracene and 12-O-tetradecanoylphorbol-13-acetate Induced Two-stage Skin Carcinogenesis

Opuntia humifusa, member of the Cactaceae family, was previously demonstrated to have radical scavenging, anti-inflammatory and anti-proliferative effects in in vitro models. It was suggested that O. humifusa could function in the prevention of carcinogenesis. To investigate the in vivo chemopreventive effect of O. humifusa, mice were fed a diet containing either 1% or 3% following 7, 12-dimethylbenz[a] anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA) induction of skin carcinogenesis. Significant decrease in the numbers of papilloma and epidermal hyperplasia were observed in mice fed with O. humifusa, compared to the control group. O. humifusa also upregulated high total antioxidant capacity and level of phase II detoxifying enzyme such as superoxide dismutase and glutathione S-transferase activity in the skin. Lipid peroxidation activity level was measured in skin cytosol and significantly inhibited in 3% OH fed group compared to the control group. These results suggest that O. humifusa exerts chemopreventive effects on chemical carcinogenesis in mouse skin and that prevention effects are associated with reduction of oxidative stress via the modulation of cutaneous lipid peroxidation, enhancing of total antioxidant capacity especially in phase II detoxifying enzyme system and partial apoptotic influence.

Tetraspanin CD151 plays a key role in skin squamous cell carcinoma.

Here we provide the first evidence that tetraspanin CD151 can support de novo carcinogenesis. During two-stage mouse skin chemical carcinogenesis, CD151 reduces tumor lag time and increases incidence, multiplicity, size, and progression to malignant squamous cell carcinoma (SCC), while supporting both cell survival during tumor initiation and cell proliferation during the promotion phase. In human skin SCC, CD151 expression is selectively elevated compared to other skin cancer types. CD151 support of keratinocyte survival and proliferation may depend on activation of transcription factor STAT3, a regulator of cell proliferation and apoptosis. CD151 also supports PKCα-α6β4 integrin association and PKC-dependent β4 S1424 phosphorylation, while regulating α6β4 distribution. CD151-PKCα effects on integrin β4 phosphorylation and subcellular localization are consistent with epithelial disruption to a less polarized, more invasive state. CD151 ablation, while minimally affecting normal cell and normal mouse functions, markedly sensitized mouse skin and epidermoid cells to chemicals/drugs including DMBA (mutagen) and camptothecin (topoisomerase inhibitor), as well as to agents targeting EGFR, PKC, Jak2/Tyk2, and STAT3. Hence, CD151 ‘co-targeting’ may be therapeutically beneficial. These findings not only support CD151 as a potential tumor target, but also should apply to other cancers utilizing CD151-laminin-binding integrin complexes.

Photocarcinogenesis and toxicity of benzoyl peroxide in hairless mice after simulated solar radiation

Topical benzoyl peroxide (BPO) gel has long been used to treat acne vulgaris and has recently been combined with clindamycin (BPO-clin). No skin malignancies have been reported after clinical use of BPO, but there has been concern about the possible carcinogenicity of BPO alone and in combination with UV radiation. BPO can promote skin tumorigenesis in a mouse skin chemical carcinogenesis model. As acne vulgaris is frequently localized on sun-exposed areas, we investigated whether BPO or BPO-clin accelerates photocarcinogenesis in combination with simulated solar radiation (SSR) in 12 groups of 25 hairless female C3.Cg/TifBomTac-immunocompetent mice. BPO or BPO-clin was applied topically to the back five times each week, followed by SSR three times each week (2, 3, or 4 standard erythema doses) 3-4 h later, for 365 days or until death. Generally BPO and BPO-clin did not accelerate the time to first, second or third tumor. Therefore, there is no evidence suggesting that BPO or BPO-clin is photocarcinogenic. However, we found significantly higher mortality in the SSR exposed groups receiving BPO and BPO-clin compared with groups receiving only BPO or BPO-clin. Our results show that BPO and the combination of BPO and clindamycin do not accelerate photocarcinogenesis, but are toxic in hairless mice. Based on the current data, the cancer risk associated with the use of BPO and BPO-clin in sun-exposed areas is minimal. Thus, while the carcinogenic potential of BPO is not fully understood, at the present time, evidence suggests that this compound is safe to use.

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.

Dietary grape seed proanthocyanidins inhibit 12-O-tetradecanoyl phorbol-13-acetate-caused skin tumor promotion in 7,12-dimethylbenz[a]anthracene-initiated mouse skin, which is associated with the inhibition of inflammatory responses

Grape seed proanthocyanidins (GSPs) possess anticarcinogenic activities. Here, we assessed the effects of dietary GSPs on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin tumor promotion in 7,12-dimethylbenz[a]anthracene (DMBA)-initiated mouse skin. Administration of dietary GSPs (0.2 and 0.5%, wt/wt) supplemented with control AIN76A diet resulted in significant inhibition of TPA-induced skin tumor promotion in C3H/HeN mice. The mice treated with GSPs developed a significantly lower tumor burden in terms of the percentage of mice with tumors (P < 0.05), total number of tumors per group (P < 0.01, n = 20) and total tumor volume per tumor-bearing mouse (P < 0.01-0.001) as compared with the mice that received the control diet. GSPs also delayed the malignant progression of papillomas into carcinomas. As TPA-induced inflammatory responses are used routinely as markers of skin tumor promotion, we assessed the effect of GSPs on biomarkers of TPA-induced inflammation. Immunohistochemical analysis and western blotting revealed that GSPs significantly inhibited expression of cyclooxygenase-2 (COX-2), prostaglandin E(2) (PGE(2)) and markers of proliferation (proliferating cell nuclear antigen and cyclin D1) in both the DMBA-initiated/TPA-promoted mouse skin and skin tumors. In short-term experiments in which the mouse skin was treated with acute or multiple TPA applications, we found that dietary GSPs inhibited TPA-induced edema, hyperplasia, leukocytes infiltration, myeloperoxidase, COX-2 expression and PGE(2) production in the mouse skin. The inhibitory effect of GSPs was also observed against other structurally different skin tumor promoter-induced inflammation in the skin. Together, our results show that dietary GSPs inhibit chemical carcinogenesis in mouse skin and that the inhibition of skin tumorigenesis by GSPs is associated with the inhibition of inflammatory responses caused by tumor promoters.

Stage-specific disruption of Stat3 demonstrates a direct requirement during both the initiation and promotion stages of mouse skin tumorigenesis

Constitutive activation of signal transducer and activator of transcription 3 (Stat3) has been found in a variety of human malignancies and has been suggested to play an important role in carcinogenesis. Recently, our laboratory demonstrated that Stat3 is required for the development of skin tumors via two-stage carcinogenesis using skin-specific loss-of-function transgenic mice. To investigate further the role of Stat3 in each stage of chemical carcinogenesis in mouse skin, i.e. initiation and promotion stages, we generated inducible Stat3-deficient mice (K5.Cre-ER(T2) x Stat3(fl/fl)) that show epidermal-specific disruption of Stat3 following topical treatment with 4-hydroxytamoxifen (TM). The epidermis of inducible Stat3-deficient mice treated with TM showed a significant increase in apoptosis induced by 7,12-dimethylbenz[a]anthracene (DMBA) and reduced proliferation following exposure to 12-O-tetradecanoylphorbol-13-acetate. In two-stage skin carcinogenesis assays, inducible Stat3-deficient mice treated with TM during the promotion stage showed a significant delay of tumor development and a significantly reduced number of tumors compared with control groups. Inducible Stat3-deficient mice treated with TM before initiation with DMBA also showed a significant delay in tumor development and a significantly reduced number of tumors compared with control groups. Finally, treatment of inducible Stat3-deficient mice that had existing skin tumors generated by the two-stage carcinogenesis protocol with TM (by intraperitoneal injection) led to inhibition of tumor growth compared with tumors formed in control groups. Collectively, these results directly demonstrate that Stat3 is required for skin tumor development during both the initiation and promotion stages of skin carcinogenesis in vivo.

Polymeric black tea polyphenols inhibit mouse skin chemical carcinogenesis by decreasing cell proliferation

The aim of this study was to investigate the antitumour promoting effects and possible mechanisms of action of the most abundant polymeric black tea polyphenols (PBPs 1-5) or thearubigins, in vivo. Effect of PBP pre-treatments on 12-O-tetradecanoylphorbol-13-acetate (TPA) promoted skin papillomas was studied in 7,12-dimethylbenz(a)anthracene initiated mice over 40 weeks. Cell proliferation and apoptosis, in epidermis of the skin, were measured using appropriate immunohistochemical staining. Mitogen-activated protein kinase signalling studies were conducted with Western blot analysis at 10, 20, 30 and 40 weeks of promotion. Pre-treatments with PBP fractions differentially altered latency, multiplicity and incidence of skin papillomas as compared to TPA treatments thereby exhibiting antipromoting effects. Most PBP fractions decreased TPA-induced cell proliferation by decreasing activation of signalling kinases (c-Jun N-terminal protein kinase, extracellular signal-regulated protein kinase, p38 protein kinase and Akt), transcription factors (activator protein-1 and nuclear factor kappa B) and inflammatory protein (cyclooxygenase 2). TPA-induced epidermal cell apoptosis was also decreased by pre-treatment with most PBP fractions. Higher levels of p53 and p21 in skin cells pre-treated with PBP fractions followed by TPA treatment as compared to only TPA-treated animals suggested possible activation of a cell cycle checkpoint. PBP-2 was observed to be the most potent polymeric polyphenol fraction and PBP-4 and PBP-5 showed only marginal activity, whereas PBP-1 and PBP-3 displayed intermediate efficacies. In conclusion, the protective effects of PBP fractions could be attributed to inhibition of TPA-induced cellular proliferation.

An action spectrum (290–320 nm) for TNFα protein in human skinin vivosuggests that basal-layer epidermal DNA is the chromophore

Terrestrial solar UVB radiation ( approximately 295-320 nm) readily induces cyclobutane pyrimidine dimers (CPDs) in human skin DNA that result in characteristic mutations associated with nonmelanoma skin cancer. The proinflammatory cytokine TNFalpha is important in mouse skin chemical carcinogenesis and is thought to also play a role in UVR-induced skin cancer by its immunomodulatory properties. There is some in vitro evidence that CPDs initiate the production of TNFalpha, and we tested this hypothesis by comparing the wavelength dependence (action spectrum) for TNFalpha protein induction in human skin in vivo with our earlier in vivo action spectra for CPD induction in four different epidermal layers of human skin. Normal volunteers (n = 35) were irradiated with physiologically relevant doses of monochromatic UVB (290-320 nm), and TNFalpha concentration was assessed, by high-sensitivity ELISA, in exudates from skin suction blisters raised 8 h after irradiation. An action spectrum, constructed from the slopes of the dose-response curves at the different wavelengths, showed maximal efficacy at 300 nm. An excellent match was observed for TNFalpha and the CPD action spectrum for cells in the lower basal epidermis. These data strongly suggest that UVB-induced photodamage to DNA in the epidermal basal layer is a major trigger for TNFalpha production. The TNFalpha may originate directly from the keratinocytes in this layer or inflammatory cells that are rapidly recruited into the upper dermis (e.g., neutrophils) as a consequence of DNA photodamage to basal-layer keratinocytes.

Cutaneous Two-Stage Chemical Carcinogenesis

INTRODUCTIONThe induction of cutaneous carcinogenesis in mice by topical administration of chemicals enables the local, systemic, and environmental factors that influence tumor susceptibility, growth, and progression to be studied in the laboratory. Chemical carcinogenesis in mouse skin has been used for several decades, and continues to help in the identification of important molecular and immunological pathways involved in cutaneous malignancy. This protocol describes the two-stage application of chemicals to the skin for the initiation and promotion of cutaneous tumors. In the two-stage model of induction, a single application of the chemical initiator mutagen 7,12-dimethylbenz[a]anthracene (DMBA) is followed by repeated applications of a pro-inflammatory phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA; otherwise known as phorbol 12-myristate 13-acetate [PMA]). Resulting tumors may develop in the form of benign papillomas that regress or progress to SCC, or SCC may arise without an apparent precursor lesion. Thus, two-stage chemical carcinogenesis provides an opportunity to monitor early and late events in cancer development and progression.

Retinoids and skin: Microarrays shed new light on chemopreventive action of all‐trans retinoic acid

Despite the use of retinoids in the clinic for many years, their mode of action in the prevention of skin cancer is still unclear. Recent microarray analyses of the chemopreventive effect of all-trans retinoic acid (ATRA), one of the primary naturally occurring biologically active retinoids, in the two-stage mouse skin chemical carcinogenesis model have provided novel insight into their action. Comparison of the gene expression profiles of control skin to skin subjected to the two-stage protocol for 3 wk, with or without ATRA, has shown that approximately half of the genes regulated by 12-o-tetradecanoylphorbol-13-acetate (TPA) are oppositely regulated when ATRA is coadministered with TPA. It was further shown the Raf/Mek/Erk branch of mitogen-activated protein (MAP) kinase pathway contains a disproportionate number of oppositely regulated genes, thereby implicating it as one of the key pathways involved in tumor promotion by TPA, that is blocked by ATRA. This result has pointed the way toward the detailed study of Raf/Mek/Erk pathway signaling in skin cancer development and its potential as a target pathway for chemoprevention by ATRA and other chemopreventive drugs.

Mouse skin chemical carcinogenesis is inhibited by antizyme in promotion‐sensitive and promotion‐resistant genetic backgrounds

Elevated polyamine content and increased ornithine decarboxylase (ODC) activity have been associated with neoplastic growth in numerous animal models and human tissues. Antizyme (AZ) is a negative regulator of polyamine metabolism that inhibits ODC activity, stimulates ODC degradation, and suppresses polyamine uptake. Preliminary evidence, obtained from transgenic mice with tissue specific overexpression of AZ indicates that tumor development can be suppressed by AZ. To extend these studies, we have examined the effect of keratin 5 (K5)- or K6-driven AZ transgenes on 7,12-dimethylbenz[a]anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) chemical carcinogenesis of the skin, in promotion-resistant C57BL/6 and promotion-sensitive DBA/2 mice. On both genetic backgrounds, K6-AZ mice showed a reduction in tumor multiplicity, with 85% fewer tumors than wild-type controls on the C57BL/6 background and 50% fewer tumors on the DBA/2 background. K5-AZ mice developed 50% fewer tumors than controls on both backgrounds. The percent of mice with tumors and tumor size were also reduced in the K5-AZ and K6-AZ groups. Tumor and TPA-treated skin sections from K6-AZ mice exhibited the strongest AZ expression, with localization mainly in suprabasal keratinocytes. K6-AZ mice also had slightly reduced cell proliferation rates in tumors and TPA-treated skin. The lack of a more pronounced effect on cell proliferation is probably explained by the observation that AZ staining did not colocalize with proliferating cell nuclear antigen (PCNA), a marker for the proliferative compartment. These studies demonstrate a tumor-suppressive effect of AZ in C57BL/6 and DBA/2 mice, and confirm the importance of ODC and polyamines in tumor development.

Genetic analyses of mouse skin tumor progression susceptibility using SENCAR inbred derived strains.

Susceptibility to tumor development varies among individuals in the human population. This variability can also be found among different strains of mice, particularly in the mouse skin chemical carcinogenesis model. The genetic mechanisms underlying mouse skin tumor susceptibility are not fully understood. The SENCAR stock has been found to be the most sensitive mice for skin carcinogenesis studies; however, little is known about the genes underlying tumor susceptibility, particularly, those involved in tumor progression. Experiments with the SSIN/Sprd mice, an inbred strain derived from the outbred SENCAR stock, suggested that papilloma development, tumor promotion, and their conversion into squamous cell carcinomas (SCCs), progression, are regulated by different genes. In the highly sensitive SSIN/Sprd mice, papillomas rarely progress to SCC. Using crosses between the outbred SENCAR and the SSIN/Sprd mice, we previously determined that papilloma progression in the SENCAR stock could be controlled by at least one autosomal dominant gene. However, the outbred nature of the SENCAR stock precluded us from extending those findings. More recently, another inbred strain was developed from the outbred SENCAR stock, the SENCARB/Pt. These mice have similar tumor promotion sensitivity to the SSIN/Sprd but in contrast, have high papilloma progression susceptibility, similar to the outbred original stock. In the present study, we generated F(1), F(2), and backcross hybrids between the SSIN/Sprd and SENCARB/Pt mice to determine a possible model for tumor progression susceptibility and to map the putative tumor susceptibility genes. Our tumor data suggests that papilloma progression susceptibility in the SENCAR mouse skin model could be genetically determined by one susceptibility gene. Our preliminary linkage analysis failed to identify one strong susceptibility locus to confirm this but provided some evidence for at least one possible susceptibility locus in mouse chromosome 14.

Loss of JunB activity enhances stromelysin 1 expression in a model of the epithelial-to-mesenchymal transition of mouse skin tumors.

Chemical carcinogenesis in mouse skin has been useful in delineating the molecular events that underlie squamous cell carcinoma progression. A late event in this progression, the epithelial-to-mesenchymal transition (EMT), is characterized by the loss of epithelial markers and the presence of mesenchymal markers. One mesenchymal marker associated with this transition is the matrix metalloproteinase stromelysin 1 (Str-1). To examine the molecular mechanisms regulating the expression of Str-1 during the EMT, genetically related mouse skin tumor cell lines representing the epithelial (B9(SQ)) and mesenchymal (A5(SP)) phenotypes were studied. As expected, B9(SQ) cells did not make Str-1, while A5(SP) cells did. B9(SQ)-A5(SP) somatic hybrids did not make Str-1, suggesting that a critical regulatory factor was a B9(SQ)-specific repressor. Str-1 promoter analysis revealed that a canonical AP-1 site was sufficient to maintain differential reporter gene activity. This result correlated with the observed loss of binding of the transcriptionally inactive JunB-Fra-2 AP-1 complex from B9(SQ) cells, being replaced primarily by the more active JunD-Fra-2 complex in A5(SP) cells. The higher level of JunB binding to both DNA and Fra-2 correlated with its hyperphosphorylation by Jun N-terminal kinase, an activity that was significantly higher in B9(SQ) cells. In the somatic hybrids, JunB gene expression was highly upregulated, a condition that also was sufficient to repress the expression of the endogenous Str-1 gene in A5(SP) cells. These data suggested that alterations in JunB activity, by changes in either phosphorylation or gene expression, contributed to the phenotypic differences that occur in this model of the EMT.

Increased apoptosis during papilloma development in mice susceptible to tumor progression.

Programmed cell death (apoptosis) is known to occur not only during normal development and tissue remodeling but also during neoplasia. Despite the suggested role of apoptosis in preventing the proliferation of malignant cells, a positive correlation between tumor progression and the presence of apoptotic cells has been found in different types of cancer, including epithelial tumors. In normal mouse skin, the role of apoptosis is not completely understood, and it has been suggested that terminal differentiation may be a special case of apoptosis. In the work reported here, we counted apoptotic cells in mouse skin tumors generated with a two-stage chemical carcinogenesis protocol. We analyzed papillomas from outbred SENCAR mice at different times during promotion, and to better determine the correlation between apoptosis and tumor progression, we compared papillomas generated from two inbred strains derived from the SENCAR stock that differ in their susceptibility to tumor progression. Our results showed that in mouse skin chemical carcinogenesis, the number of apoptotic cells was greater in papillomas that may have been in the process of progressing to squamous cell carcinomas. This conclusion is also supported by the fact that papillomas from SENCAR P/Bt. mice, a tumor progression-susceptible strain derived from outbred SENCAR mice, had more apoptotic cells than papillomas from progression-resistant SSIN mice.

Protective effect of all trans retinoic acid against tumor promotion and progression in low- and high-risk protocols of mouse skin chemical carcinogenesis

In this study, we assessed the protective effects of all trans retinoic acid (RA) against skin tumor promotion and progression in three different protocols of mouse skin multistage carcinogenesis. Under these protocols, where papillomas on the skin of SENCAR mice were induced for their low- and high-probability of conversion to malignant carcinomas, pre-application of RA (10 mu g/mouse/application) to that of tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or mezerein (MEZ) in 7,12-dimethylbenz(a)anthracene (DMBA)-initiated mouse skin resulted in a highly significant protection against skin tumor promotion. In terms of tumor incidence, multiplicity and volume/mouse, at the termination of the experiment at 20 weeks, RA showed 35-80%, 67-83% and 70-98% protection, respectively. While the effect of RA was profound in all the protocols, maximum affectivity was evident in high-risk papilloma induction protocol where DMBA was used as tumor initiator and one week later TPA was applied once a week for five weeks. In tumor progression studies, at 20 weeks of standard low- and high-risk protocols, when papilloma yield is stabilized, animals which did not receive RA in different protocols were divided into two groups and topically treated twice weekly either with acetone or RA (10 mu g). These treatments were continued for an additional 29 weeks. During these treatments, all suspected carcinoma formation was recorded and carcinomas were verified histologically at the termination of the experiment. In each case, RA showed remarkable protective effects against percentage of mice with carcinomas (35-45% protection), number of carcinomas per mouse (50-63% protection) and the rate of malignant conversion (50-63% protection). The results of the present study clearly demonstrate that irrespective of the risk for skin carcinogenesis, RA is capable of affording protection against the induction of nonmalignant lesions and their subsequent conversion to malignancy.

Role of vitamin D3 on the activity patterns of hepatic drug metabolizing enzymes in transplantable murine lymphoma.

Vitamin D3 (D3) has been found to exert varied pharmacological actions including restriction of cell growth of a number of malignant cell lines in vitro and inhibition of the promotion of chemical carcinogenesis in mouse skin. In an attempt to confirm the efficacy of D3 as an antineoplastic agent, the present investigation aims at characterizing the importance of D3 in modulating hepatic drug metabolizing enzymes, namely, cytosolic glutathione S-transferase (GSHT), microsomal UDP glucuronyl transferase (UDPGT), and cytochrome P-450, which have been reported by us in recent literature as significant neoplastic markers in mice bearing Dalton's lymphoma (DL). Results show that D3 causes a 150% elevation of GSHT activity and the maintenance of normal, near-control UDPGT activity and cytochrome P-450 content, up to almost 30 days following tumor transplantation, along with bringing about a twofold increase in survival of the host mice. In conclusion, we confirm the definite and significant antitumorigenic role of D3 and its involvement with the discussed hepatic tumor markers in monitoring the processes that lead to cell survival.

Transgenic approaches to understanding the mechanisms of chemical carcinogenesis in mouse skin

The use of animal models for human cancer has proved effective in the elucidation of those molecular events which are responsible for the various stages of tumour development. Chemical carcinogenesis in mouse skin has been studied as a model for human squamous cancer for several decades, and analysis of this model has led to the identification of a number of the changes which are involved in the evolution of malignancy. The use of transgenic and knockout mice offers a further avenue of advancement, allowing refinement of the model, and the ability to examine the consequences of individual events in vivo in greater detail. Additionally, crossing different transgenic or knockout animals represents a powerful tool to study the cumulative effects of several genetic alterations acting in concert.

Immunohistochemical characterization of cellular infiltrates in epidermal tumors induced by two-stage and complete chemical carcinogenesis in mouse skin.

We investigated the population and pattern of the infiltrated cells in both benign and malignant epidermal tumors which were induced chemically with benzo(a)pyrene (B(a)P) in murine skin. In benign papillomas, which were evolved by a two stage carcinogenesis regimen, a slight to mild inflammatory infiltration around the tumors was observed, and cells infiltrating into the tumor nests were rarely seen. In carcinomas, which were produced by a complete carcinogenesis regimen, a dense inflammatory infiltration was observed around the tumor nests. The infiltrated cells were characterized as T-lymphocytes, macrophages, and neutrophils. Natural killer (NK) cells were found around and in the tumor nests, but their number was small. Both T-lymphocytes and macrophages were found to invade the tumor nests in squamous cell carcinoma whose duration was more than four weeks. This experimental carcinogenesis animal model allows the detailed quantitative and functional analysis of the infiltration of immunocompetent cells into epidermal tumors.