Mouse Epidermal Tumors Research Articles

The downregulation of ΔNp63 in p53-deficient mouse epidermal tumors favors metastatic behavior.

The TP63 gene codes for two major isoform types, TAp63 and ΔNp63, with probable opposite roles in tumorigenesis. The ΔNp63α protein is frequently amplified and overexpressed in different epithelial tumors. Accordingly, it has been considered a potential oncogene. Nonetheless, a possible metastatic suppressor activity has also been suggested based on the experimental observation that its expression is reduced or even absent in advanced invasive tumors. Such metastatic suppressor activities are often related to tumors bearing point mutated TP53 gene. However, its potential roles in TP53-deficient tumors are poorly characterized. Here we show that in spontaneous tumors, induced by the epidermal-specific Trp53 ablation, the reduction of ΔNp63 expression is an early event, whereas it is re-expressed in the lung metastatic lesions. Using knock down and ectopic expression approaches, we show that ΔNp63 expression opposes the epithelial-mesenchymal transition and reduces the metastatic potential of the cells. This process occurs through the modulation of ΔNp63-dependent downstream targets (including transcription factors and microRNAs) likely to play metastatic roles. Further, ΔNp63 also favors the expression of factors involved in iPS reprogramming, thus suggesting that it can also modulate specific stem cell traits in mouse epidermal tumor cells. Overall, our data assign antimetastatic roles to ΔNp63 in the context of p53 deficiency and epidermis.

Conjugated Linoleic Acid and Disease Prevention: A Review of Current Knowledge

Conjugated linoleic acid (CLA), a derivative of a fatty acid linoleic acid (LA), has been reported to decrease tumorigenesis in animals. CLA is unique because unlike most antioxidants which are components of plant products, it is present in food from animal sources such as dairy foods and meats. CLA concentrations in dairy products typically range from 2.9 to 8.92 mg/g fat of which the 9-cis, 11-trans isomer makes up to 73% to 93% of the total CLA. Low concentrations of CLA are found in human blood and tissues. In vitro results suggest that CLA is cytotoxic to MCF-7 cells and it inhibits the proliferation of human malignant melanoma and colorectal cancer cells. In animal studies, CLA has inhibited the development of mouse epidermal tumors, mouse forestomach cancer and rat mammary cancer. Hamsters fed CLA collectively had significantly reduced levels of plasma total cholesterol, non-high-density lipoprotein cholesterol, (combined very-low and low-density lipoprotein) and triglycerides with no effect on high-density lipoprotein cholesterol, as compared to controls. Dietary CLA modulated certain aspects of the immune defense but had no obvious effect on the growth of an established, aggressive mammary tumor in mice. It is now thought that CLA itself may not have anti-oxidant capabilities but may produce substances which protect cells from the detrimental effects of peroxides.There is, however, insufficient evidence from human epidemiological data, and very few of the animal studies have shown a dose-response relationship with the quantity of CLA feed and the extent of tumor growth. Further research with tumor models is needed to test the efficacy and utility of CLA in cancer and other disease prevention and form the basis of evaluating its effect in humans by observational studies and clinical trials.

Augmented expression of cytokines in mouse epidermal tumor cells and its possible involvement in the induction of hematopoietic alterations

Mice with skin tumors induced either by 7,12-dimethylbenz[a]anthracene complete carcinogenesis or subcutaneous injection of a carcinogenic keratinocyte cell line showed moderate to severe splenomegaly as a result of an increase in splenic granulocyte-macrophage and erythroid (erythroid burst-forming unit) progenitors. To test whether the observed alterations involve the release of soluble factors by the epidermal component of skin tumors, we used an in vitro approach. A series of mouse keratinocyte cell lines resembling progressive stages of skin carcinogenesis and carrying either normal or activated Ha-ras genes were assayed for their ability to produce the factors required for colony growth of hematopoietic-committed progenitors. Only the conditioned media of keratinocytes harboring activated Ha-ras genes were able to support the growth of granulocyte-macrophage colony-forming units. In addition, preincubation of normal bone-marrow cells with conditioned media from the transformed epidermal cell lines stimulated in vitro amplification of the hematopoietic granulocyte-macrophage progenitor compartment. To identify the possible factors responsible for the activities detected in the keratinocyte-conditioned media, we performed northern blot analysis using the cytokine probes granulocyte colony-stimulating factor, macrophage colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, stem cell factor, interleukin-1 alpha, interleukin-3, and tumor necrosis factor-alpha. The cell lines expressed different cytokine mRNA combinations that positively correlated with the colony-stimulating activity detected in the corresponding conditioned medium. These results suggest that transformed epidermal tumor cells in vivo may alter normal hematopoiesis as a consequence of the production of cytokines that act in autocrine or paracrine loops probably related to tumor growth.

Changes in Keratin Expression during Malignant Progression of Transformed Mouse Epidermal Keratinocytes

We have studied in this report the expression of keratins in mouse epidermal keratinocytes transformed in culture by a chemical carcinogen (PDV) and in cell lines (PDVCM1, PDVCM2, PDVCV1, and PDVC57) derived by tumor transplantation of PDV cells in syngeneic C57BI/6 mice. PDV, PDVCM1, PDVCM2, and PDVCV1 cell lines are weakly to moderately tumorigenic, giving rise to squamous cell carcinomas, although not at all injection points, whereas PDVC57 cells are more malignant, inducing highly anaplastic carcinomas at 100% of injection sites. All the cell lines synthesize anomalously simple epithelial keratins, substantial amounts of K8, and minor quantities of K18 and K19, but the level of expression is increased in PDVC57. We have found that in PDVC57 cells upregulation of K8 is linked to down-regulation of the normal keratins produced by epidermal keratinocytes in culture (i.e., K5, K6, K14, and K17). On the other hand, K8 does not generally colocalize with K13, a keratin also aberrantly expressed by epidermal cell cultures when induced to differentiate by high Ca 2+ medium. K13, normally synthesized in internal stratified epithelia, is anomalously induced in mouse epidermal tumors and has been used as an early marker of carcinoma progression. In tumors induced by the cell lines upon injection in mice, K8 is found in the less differentiated regions as opposite to K13, restricted to the differentiating areas of the tumors. In PDV, PDVCM1, PDVCM2, and PDVCV1 carcinomas the overall expression of K13 is higher than that of K8. However, this relation is inverted in tumors induced by PDVC57 cells, in a good correlation with the tumoral phenotypes produced by the cell lines. Our results suggest that upregulation of the simple epithelial keratin K8, as found in transformed epidermal cell lines and tumors, is a late marker of malignant progression and is associated with the loss of the differentiated phenotype.

Inhibition of tumor formation from grafted murine papilloma cells by treatment of grafts with staurosporine, an inducer of squamous differentiation.

The microbial alkaloid staurosporine induces responses associated with protein kinase C activation, resulting in terminal differentiation in cultures of both normal and neoplastic mouse epidermal cells. As a cancer chemotherapy model, we treated grafts of mouse epidermal tumor cell lines 308 and SP-1 repeatedly with staurosporine. A dose-dependent inhibition of tumor formation, maximal at 0.025 nmol per treatment, was observed. Higher and lower doses were less effective, suggesting a specific target for staurosporine action. A single, low-dose treatment 2 weeks after grafting also markedly reduced tumor formation. Although in vitro evidence suggests that staurosporine-induced terminal squamous differentiation results from activation of protein kinase C, we found no inhibition of tumor growth in similar studies with the protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate. These results indicate that staurosporine is an effective antitumor agent for eradicating squamous cell tumors in vivo.

Aberrant expression of the simple epithelial type II keratin 8 by mouse skin carcinomas but not papillomas.

Keratins have been demonstrated to be suitable markers of changes taking place during epithelial neoplasia. Therefore, we analyzed 18 mouse skin tumors (nine papillomas and nine squamous cell carcinomas), induced either by two-stage carcinogenesis with 7,12-dimethylbenz[a]anthracene(DMBA)/12-O-tetradecanoylphorbol-13-acetat e or complete carcinogenesis with DMBA, by immunofluorescence with a monoclonal antibody to keratin (K) 8 (TROMA-1). Immunoperoxidase staining and immunoblotting were also used on selected tumor samples to further explore for the presence of K8. All of the papillomas tested were negative for the presence of K8, whereas the carcinomas were positive. The level of K8 expression in carcinomas showed a positive correlation with the degree of malignancy. Northern blot analysis using a K8 cDNA probe suggested that control of K8 expression in mouse skin tumors occurs at the transcriptional level. Double-label immunofluorescence staining using TROMA-1 and RK13 antibodies demonstrated that K8 did not generally colocalize with K13, a keratin normally found in internal stratified epithelial but aberrantly expressed in mouse epidermal tumors. Furthermore, tumors expressing high levels of K8 showed a reduced expression of K13. Histological examination of immunoperoxidase-stained tumors demonstrated that K8-positive cells were mainly found in anaplastic areas, whereas K13 foci were restricted to well-differentiated regions. Our results demonstrate that K8 expression is a marker of late stages of carcinoma progression in the mouse skin carcinogenesis model.

V‐Ha‐ras‐induced mouse skin papillomas exhibit aberrant expression of keratin K13 as do their 7,12‐dimethylbenz[a]anthracene/12‐O‐tetradecanoylphorbol‐13‐acetate‐induced analogues

Introduction of the v-Ha-ras gene into primary epidermal keratinocytes, followed by grafting of these cells to animals, leads to the formation of benign epidermal tumors that resemble papillomas induced chemically by a two-stage carcinogenesis protocol. In this study, we investigated v-Ha-ras-induced papillomas for aberrant expression of type I keratin K13, previously described in 7,12-dimethylbenz[a]anthracene/12-O-tetradecanoylphorbol-13- acetate (DMBA/TPA)-induced mouse epidermal tumors. Papillomas produced from three independent infection series were removed 3 wk after grafting concomitant with control grafts originating from mock-, neo-, and v-fos-infected primary keratinocytes. Combined analysis of the grafts by western blotting of extracted keratins and immunofluorescence studies of frozen sections with a K13-monospecific antibody revealed K13 expression in all v-Ha-ras-induced papillomas and absence of this keratin in all control grafts. K13-positive cells in papillomas were restricted to the suprabasal cell layers of the lesions and, at this stage of papilloma development, occurred as foci of varying extensions. Analysis of genomic DNA from v-Ha-ras-induced papillomas for the methylation state of a CpG dinucleotide in the distant promoter region of the K13 gene revealed the occurrence of unmethylated DNA copies that were generated at the expense of methylated DNA copies ubiquitously present in normal epidermis. The ratio of unmethylated to methylated DNA copies correlated with the extent of suprabasal K13 protein expression. Thus, all features of aberrant K13 expression previously described in DMBA/TPA-induced papillomas were shared by v-Ha-ras-induced papillomas.

Presence of a functionally altered ornithine decarboxylase activity in chrysarobin-promoted mouse epidermal papillomas.

Recent work from this laboratory has demonstrated the presence of a structurally and functionally different ornithine decarboxylase (ODC) in mouse epidermal tumors induced by a two-stage protocol involving initiation with 7,12-dimethylbenzanthracene (DMBA) and promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA). In this report, the enzymatic properties of ODC present in DMBA-initiated chrysarobin-promoted papillomas are compared to the enzyme induced by chrysarobin in normal epidermis. Analyses of 13 individual tumor extracts indicated each had an elevated level of ODC activity compared to uninduced normal epidermis. Addition of GTP to the enzyme assay caused a marked stimulation of ODC activity in nine of 13 tumor extracts but had no effect on chrysarobin-induced epidermal ODC. Enzyme kinetic analyses indicated that GTP lowered the atypically high apparent Km values for L-ornithine of the papilloma enzyme to values typical of epidermal ODC. The K 1/2 for GTP activation of papilloma ODC was approximately 7 x 10(-9) M. When a series of nucleotides was tested, only GTP, the non-hydrolysable analog GTP gamma S, dGTP and GDP were capable of significant activation at 1 microM, while other derivatives including GMP, ATP and CTP were less effective. The ability of the tumor enzyme to bind GTP was confirmed by the results of GTP-agarose chromatography, in which the papilloma enzyme (but not chrysarobin-induced epidermal ODC) bound to this affinity column and could be eluted by GTP. While some differences were observed in the properties of ODC from chrysarobin-promoted versus TPA-promoted papillomas, the major conclusion of this study is that both agents cause the appearance of a functionally altered ODC in the majority of papillomas produced by a two-step protocol.

Activation of mouse epidermal tumor ornithine decarboxylase by GTP: evidence for different catalytic forms of the enzyme.

In crude extracts of epidermal papillomas induced by an initiation-promotion protocol, ornithine decarboxylase (OrnDCase) activity was increased by the addition of GTP to the enzyme assay. No effect of GTP on the phorbol ester-induced enzyme isolated from normal epidermis was observed. Kinetic analyses indicated that the major effect of the nucleotide on the tumor-derived enzyme was to lower the apparent Km for L-ornithine. When papilloma OrnDCase was partially purified by gel-filtration chromatography, two forms of the enzyme were resolved, only one of which was found in an epidermal extract from phorbol 12-myristate 13-acetate-treated mice. The enzymatic properties of the two forms of papilloma enzyme were compared. The higher molecular weight form (peak I) was activated by GTP, while the lower molecular weight form (peak II) was not. As expected from the kinetic analyses of the crude papilloma extracts, the apparent Km of peak I enzyme for L-ornithine was very high (1.25 mM) but was much lower in the presence of GTP (0.02 mM). The two forms of papilloma OrnDCase differed in their sensitivities to heat inactivation and the ability of GTP to protect against heat inactivation. The K1/2 for activation of peak I OrnDCase by GTP was 0.1 microM. The activation process was irreversible and did not require Mg2+. When several nucleotides were tested for their ability to activate peak I OrnDCase, only GTP, dGTP, and the nonhydrolyzable derivative GTP[gamma-S] were effective, while GDP, GMP, ATP, and CTP were relatively ineffective. Our results demonstrated the existence of two forms of OrnDCase in epidermal tumor extracts, of which one can be activated by GTP and one cannot. The significance of these findings for the regulation of this enzyme in normal and tumor cells is discussed.

A transforming activity not detected by DNA transfer to NIH 3T3 cells is detected by JB6 mouse epidermal cells.

Transfection of four different mouse epidermal tumor cell DNAs into NIH 3T3 cells yielded neither morphologically altered foci nor anchorage independence. However, promotion-sensitive, but not promotion-insensitive, JB6 mouse epidermal cell lines were permissive for the expression of anchorage independence after transfection of DNA from three of these tumor cell lines. This transforming activity and the promotion-sensitive activity that confers sensitivity to promotion of transformation show differences in restriction enzyme sensitivity. In view of this difference and the differences in both recipient cells and 12-O-tetradecanoyl-phorbol-13-acetate dependence of expression, it appears that the transforming activity and the promotion-sensitive activity are specified by different genes. The JB6 promotion-sensitive cell lines may be useful for detecting and cloning transforming genes that escape detection in the NIH 3T3 cell focus assay.

A transforming activity not detected by DNA transfer to NIH 3T3 cells is detected by JB6 mouse epidermal cells.

Transfection of four different mouse epidermal tumor cell DNAs into NIH 3T3 cells yielded neither morphologically altered foci nor anchorage independence. However, promotion-sensitive, but not promotion-insensitive, JB6 mouse epidermal cell lines were permissive for the expression of anchorage independence after transfection of DNA from three of these tumor cell lines. This transforming activity and the promotion-sensitive activity that confers sensitivity to promotion of transformation show differences in restriction enzyme sensitivity. In view of this difference and the differences in both recipient cells and 12-O-tetradecanoyl-phorbol-13-acetate dependence of expression, it appears that the transforming activity and the promotion-sensitive activity are specified by different genes. The JB6 promotion-sensitive cell lines may be useful for detecting and cloning transforming genes that escape detection in the NIH 3T3 cell focus assay.

A beef-derived mutagenesis modulator inhibits initiation of mouse epidermal tumors by 7,12-dimethylbenz[a]anthracene.

Ground beef contains an organic solvent extractable mutagenesis modulator. Crude or partially-purified preparations of the activity were applied to the backs of SENCAR or CD-1 mice 5 min before application of 7,12-dimethylbenz[a]anthracene (DMBA). Controls received solvent (acetone) only prior to DMBA. Following 1-2 week intervals promotion was effected with twice-weekly applications of 12-O-tetradecanoylphorbol-13-acetate. Modulator-treated mice consistently developed fewer papillomas and exhibited lower tumor incidences than did positive control mice.

Keratins as markers of malignancy in mouse epidermal tumors.

A comparative study of the keratin composition of adult and neonatal mouse epidermis, benign and malignant tumors of mouse skin and murine epidermal cells grown in culture revealed striking differences in the keratin polypeptide patterns when analyzed by one-dimensional gel electrophoresis. Not only did the study confirm body-site specific alterations in the keratin patterns within one species, but it also demonstrated that similar to cultured epidermal cells, three malignant skin tumors investigated specifically lacked a group of keratin components with molecular weights larger than 61,000 daltons, which were invariably present in all normal and also in benign tissues. These findings offer the possibility of using keratins as molecular markers of the malignant state of epidermal cells. Two-dimensional gels of the keratin polypeptides of normal epidermis and of benign tumors exhibited spot patterns which could be divided at the 61,000 dalton level into an essentially basic subgroup, comprising the high molecular weight keratins, and an acidic subgroup including the low molecular weight components. The two uppermost proteins of cultured epidermal cells and carcinomas (molecular weights 61,000 and 58,000 daltons) belong to the acidic subgroup in normal tissues as well as in papillomas. However, in the case of malignant tumors and in vitro transformed epidermal cells they showed distinct alterations in charge in that they migrated into the more alkaline part of the gel. The number of spots appearing in the more basic region of the gel could be inversely related to the degree of differentiation exhibited by tumors or in vitro cells.