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Resistance of R-Ras knockout mice to skin tumour induction.

May U, Prince S, Vähätupa M, Laitinen AM, Nieminen K, Uusitalo-Järvinen H, Järvinen TA - Sci Rep (2015)

Bottom Line: The R-ras gene encodes a small GTPase that is a member of the Ras family.Despite close sequence similarities, R-Ras is functionally distinct from the prototypic Ras proteins; no transformative activity and no activating mutations of R-Ras in human malignancies have been reported for it.The DMBA/TPA skin tumourigenesis-model is highly dependent upon inflammation, and we found a greatly attenuated skin inflammatory response to DMBA/TPA-treatment in the R-Ras KO mice in the context of leukocyte infiltration and proinflammatory cytokine expression.

View Article: PubMed Central - PubMed

Affiliation: School of Medicine, Department of Anatomy and Cell Biology, University of Tampere, Tampere, Finland.

ABSTRACT
The R-ras gene encodes a small GTPase that is a member of the Ras family. Despite close sequence similarities, R-Ras is functionally distinct from the prototypic Ras proteins; no transformative activity and no activating mutations of R-Ras in human malignancies have been reported for it. R-Ras activity appears inhibitory towards tumour proliferation and invasion, and to promote cellular quiescence. Contrary to this, using mice with a deletion of the R-ras gene, we found that R-Ras facilitates DMBA/TPA-induced skin tumour induction. The tumours appeared in wild-type (WT) mice on average 6 weeks earlier than in R-Ras knockout (R-Ras KO) mice. WT mice developed almost 6 times more tumours than R-Ras KO mice. Despite strong R-Ras protein expression in the dermal blood vessels, no R-Ras could be detected in the epidermis from where the tumours arose. The DMBA/TPA skin tumourigenesis-model is highly dependent upon inflammation, and we found a greatly attenuated skin inflammatory response to DMBA/TPA-treatment in the R-Ras KO mice in the context of leukocyte infiltration and proinflammatory cytokine expression. Thus, these data suggest that despite its characterised role in promoting cellular quiescence, R-Ras is pro-tumourigenic in the DMBA/TPA tumour model and important for the inflammatory response to DMBA/TPA treatment.

No MeSH data available.


Related in: MedlinePlus

The inflammatory response to DMBA/TPA treatment is attenuated in the skin of R-Ras KO mice.WT and R-Ras KO mice were subjected to DMBA/TPA-induced skin carcinogenesis as described in methods. Skin samples were collected from untreated mice and from mice sacrificed at 3 h and 48 h after the second TPA application, and after 19 weeks of treatment (twice weekly). The skin samples were processed either for IHC or qPCR analysis as described in methods. Results are shown as mean ± 95% confidence intervals. Data were analysed by normality tests and unpaired two-tailed T-tests, if needed with Welch’s correction (GraphPad Prism 6). (a) qPCR analysis of relative gene expression of the cytokines IL-1α, IL-6 and IL-17A in untreated and treated WT and R-Ras KO skin. R-Ras KO mice show 3 h post 2nd TPA treatment significantly lower gene expression for IL-1α (P = 0.0176, *) and IL-6 (P = 0.0125, *) than WT mice. IL-17A gene expression is significantly reduced in R-Ras KO animals at 3 h post 2nd TPA treatment (P = 0.0322, *) and after 19 weeks of TPA treatment (P = 0.0189, *). These data are normally distributed. Animal numbers: untreated: n = 4 per strain, 3 h post 2nd TPA treatment: n = 8 per strain, 48 h post 2nd TPA treatment: n = 9 per strain, 19 weeks: n = 8 for WT and n = 6 for R-Ras KO. ND means not detected. (b) Skin sections were IHC stained for markers for dermal macrophages (F4/80), dermal neutrophils (neutrophil elastase), or epidermal and dermal T-cells (CD3), as described in methods (WT n = 6; R-Ras KO n = 6). Quantitative analysis of scanned slides were performed as described in methods (3 independent measurements per skin section, two skin sections per animal, excluding tumours). Data is expressed as % of total nuclei. Neutrophil and T-cell data are mostly normally distributed, but all becomes normally distributed if Log10 transformed. Macrophage data is normally distributed. T-tests confirmed highly significant differences between the WT and R-Ras KO mice as indicated (P < 0.0001, ****). (c) Representative photographs of leukocyte staining in WT and R-Ras KO skin. Nuclei are stained blue, and leukocytes brown. The black bar in images represents 300 μm.
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f5: The inflammatory response to DMBA/TPA treatment is attenuated in the skin of R-Ras KO mice.WT and R-Ras KO mice were subjected to DMBA/TPA-induced skin carcinogenesis as described in methods. Skin samples were collected from untreated mice and from mice sacrificed at 3 h and 48 h after the second TPA application, and after 19 weeks of treatment (twice weekly). The skin samples were processed either for IHC or qPCR analysis as described in methods. Results are shown as mean ± 95% confidence intervals. Data were analysed by normality tests and unpaired two-tailed T-tests, if needed with Welch’s correction (GraphPad Prism 6). (a) qPCR analysis of relative gene expression of the cytokines IL-1α, IL-6 and IL-17A in untreated and treated WT and R-Ras KO skin. R-Ras KO mice show 3 h post 2nd TPA treatment significantly lower gene expression for IL-1α (P = 0.0176, *) and IL-6 (P = 0.0125, *) than WT mice. IL-17A gene expression is significantly reduced in R-Ras KO animals at 3 h post 2nd TPA treatment (P = 0.0322, *) and after 19 weeks of TPA treatment (P = 0.0189, *). These data are normally distributed. Animal numbers: untreated: n = 4 per strain, 3 h post 2nd TPA treatment: n = 8 per strain, 48 h post 2nd TPA treatment: n = 9 per strain, 19 weeks: n = 8 for WT and n = 6 for R-Ras KO. ND means not detected. (b) Skin sections were IHC stained for markers for dermal macrophages (F4/80), dermal neutrophils (neutrophil elastase), or epidermal and dermal T-cells (CD3), as described in methods (WT n = 6; R-Ras KO n = 6). Quantitative analysis of scanned slides were performed as described in methods (3 independent measurements per skin section, two skin sections per animal, excluding tumours). Data is expressed as % of total nuclei. Neutrophil and T-cell data are mostly normally distributed, but all becomes normally distributed if Log10 transformed. Macrophage data is normally distributed. T-tests confirmed highly significant differences between the WT and R-Ras KO mice as indicated (P < 0.0001, ****). (c) Representative photographs of leukocyte staining in WT and R-Ras KO skin. Nuclei are stained blue, and leukocytes brown. The black bar in images represents 300 μm.

Mentions: It is an established fact that tumourigenesis in the DMBA/TPA model is highly dependent upon the induction of acute inflammation22. It has also recently been published that the R-Ras KO mouse has a reduced inflammatory response to experimental autoimmune encephalomyelitis due to increased tolerance in its immune system23. As our cellular transformation-focused investigations yielded no clue to the mechanism of resistance of the R-Ras KO mouse to DMBA/TPA-induced tumourigenesis, we decided to investigate if the R-Ras KO mouse had an attenuated skin inflammatory response to DMBA/TPA treatment. We measured the number of skin T-cells and infiltrating macrophages and neutrophils (via quantitative immunohistochemical analysis), as well as the gene expression of IL-1α, IL-6 and IL-17A in the skin (via qPCR analysis) at a variety of different time points following the initiation of DMBA/TPA treatment (3 h and 48 h post second TPA treatment, and after 19 weeks of twice-weekly TPA treatment). These cytokines were selected for study because, not only are they proinflammatory, but IL-17A in particular is known to be pro-tumourigenic in the DMBA/TPA model2425 and to work synergistically with IL-6262728. At 3 h after the second TPA treatment, the R-Ras KO mice showed significantly lower levels of gene expression for IL-1α, IL-17A and IL-6 compared to WT (P = 0.0176, P = 0.032 and P = 0.0125 respectively, Fig. 5a). Gene expression of IL-17A and IL-6 in the R-Ras KO mice remained low at all of the time points tested. The level of IL-17A expression in the R-Ras KO mice remained low even at the 19-week time point whilst it was significantly elevated in the WT mice (P = 0.0189, Fig. 5a). The leukocyte count data showed the lack of a significant increase in skin macrophage infiltration or T-cells in the R-Ras KO mice at any time point, with neutrophil infiltration in the R-Ras KO mice only apparently elevated at 48 h after the second TPA treatment (Fig. 5b). In contrast, in the WT mice there was a clear increase in T-cell and macrophage numbers, which was significantly greater than in the R-Ras KO mice at the 48-h time point (P < 0.0001 for each, Fig. 5b). At the 19-week time point the WT mice had significantly more T-cells and especially more neutrophils than the R-Ras KO mice (P < 0.0001 for each, Fig. 5b). When comparing the timing of the inflammatory differences between the WT and R-Ras KO mice, it was noted that the lack of cytokine expression in the R-Ras KO mice was actually most evident preceding, not following, leukocyte infiltration (extravasation) (Fig. 5). The leukocyte count data in Fig. 5b is expressed as % of total nuclei rather than cells per mm2 as either format gave very similar results (Supplementary Figure S5a), but the former is more reliable in histological areas that are slightly damaged. A measure of total cells per mm2 in all groups revealed that there were no differences in total cell density per mm2 dermis between the WT and R-Ras KO mice, either before or after treatment (Supplementary Figure S5b).


Resistance of R-Ras knockout mice to skin tumour induction.

May U, Prince S, Vähätupa M, Laitinen AM, Nieminen K, Uusitalo-Järvinen H, Järvinen TA - Sci Rep (2015)

The inflammatory response to DMBA/TPA treatment is attenuated in the skin of R-Ras KO mice.WT and R-Ras KO mice were subjected to DMBA/TPA-induced skin carcinogenesis as described in methods. Skin samples were collected from untreated mice and from mice sacrificed at 3 h and 48 h after the second TPA application, and after 19 weeks of treatment (twice weekly). The skin samples were processed either for IHC or qPCR analysis as described in methods. Results are shown as mean ± 95% confidence intervals. Data were analysed by normality tests and unpaired two-tailed T-tests, if needed with Welch’s correction (GraphPad Prism 6). (a) qPCR analysis of relative gene expression of the cytokines IL-1α, IL-6 and IL-17A in untreated and treated WT and R-Ras KO skin. R-Ras KO mice show 3 h post 2nd TPA treatment significantly lower gene expression for IL-1α (P = 0.0176, *) and IL-6 (P = 0.0125, *) than WT mice. IL-17A gene expression is significantly reduced in R-Ras KO animals at 3 h post 2nd TPA treatment (P = 0.0322, *) and after 19 weeks of TPA treatment (P = 0.0189, *). These data are normally distributed. Animal numbers: untreated: n = 4 per strain, 3 h post 2nd TPA treatment: n = 8 per strain, 48 h post 2nd TPA treatment: n = 9 per strain, 19 weeks: n = 8 for WT and n = 6 for R-Ras KO. ND means not detected. (b) Skin sections were IHC stained for markers for dermal macrophages (F4/80), dermal neutrophils (neutrophil elastase), or epidermal and dermal T-cells (CD3), as described in methods (WT n = 6; R-Ras KO n = 6). Quantitative analysis of scanned slides were performed as described in methods (3 independent measurements per skin section, two skin sections per animal, excluding tumours). Data is expressed as % of total nuclei. Neutrophil and T-cell data are mostly normally distributed, but all becomes normally distributed if Log10 transformed. Macrophage data is normally distributed. T-tests confirmed highly significant differences between the WT and R-Ras KO mice as indicated (P < 0.0001, ****). (c) Representative photographs of leukocyte staining in WT and R-Ras KO skin. Nuclei are stained blue, and leukocytes brown. The black bar in images represents 300 μm.
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f5: The inflammatory response to DMBA/TPA treatment is attenuated in the skin of R-Ras KO mice.WT and R-Ras KO mice were subjected to DMBA/TPA-induced skin carcinogenesis as described in methods. Skin samples were collected from untreated mice and from mice sacrificed at 3 h and 48 h after the second TPA application, and after 19 weeks of treatment (twice weekly). The skin samples were processed either for IHC or qPCR analysis as described in methods. Results are shown as mean ± 95% confidence intervals. Data were analysed by normality tests and unpaired two-tailed T-tests, if needed with Welch’s correction (GraphPad Prism 6). (a) qPCR analysis of relative gene expression of the cytokines IL-1α, IL-6 and IL-17A in untreated and treated WT and R-Ras KO skin. R-Ras KO mice show 3 h post 2nd TPA treatment significantly lower gene expression for IL-1α (P = 0.0176, *) and IL-6 (P = 0.0125, *) than WT mice. IL-17A gene expression is significantly reduced in R-Ras KO animals at 3 h post 2nd TPA treatment (P = 0.0322, *) and after 19 weeks of TPA treatment (P = 0.0189, *). These data are normally distributed. Animal numbers: untreated: n = 4 per strain, 3 h post 2nd TPA treatment: n = 8 per strain, 48 h post 2nd TPA treatment: n = 9 per strain, 19 weeks: n = 8 for WT and n = 6 for R-Ras KO. ND means not detected. (b) Skin sections were IHC stained for markers for dermal macrophages (F4/80), dermal neutrophils (neutrophil elastase), or epidermal and dermal T-cells (CD3), as described in methods (WT n = 6; R-Ras KO n = 6). Quantitative analysis of scanned slides were performed as described in methods (3 independent measurements per skin section, two skin sections per animal, excluding tumours). Data is expressed as % of total nuclei. Neutrophil and T-cell data are mostly normally distributed, but all becomes normally distributed if Log10 transformed. Macrophage data is normally distributed. T-tests confirmed highly significant differences between the WT and R-Ras KO mice as indicated (P < 0.0001, ****). (c) Representative photographs of leukocyte staining in WT and R-Ras KO skin. Nuclei are stained blue, and leukocytes brown. The black bar in images represents 300 μm.
Mentions: It is an established fact that tumourigenesis in the DMBA/TPA model is highly dependent upon the induction of acute inflammation22. It has also recently been published that the R-Ras KO mouse has a reduced inflammatory response to experimental autoimmune encephalomyelitis due to increased tolerance in its immune system23. As our cellular transformation-focused investigations yielded no clue to the mechanism of resistance of the R-Ras KO mouse to DMBA/TPA-induced tumourigenesis, we decided to investigate if the R-Ras KO mouse had an attenuated skin inflammatory response to DMBA/TPA treatment. We measured the number of skin T-cells and infiltrating macrophages and neutrophils (via quantitative immunohistochemical analysis), as well as the gene expression of IL-1α, IL-6 and IL-17A in the skin (via qPCR analysis) at a variety of different time points following the initiation of DMBA/TPA treatment (3 h and 48 h post second TPA treatment, and after 19 weeks of twice-weekly TPA treatment). These cytokines were selected for study because, not only are they proinflammatory, but IL-17A in particular is known to be pro-tumourigenic in the DMBA/TPA model2425 and to work synergistically with IL-6262728. At 3 h after the second TPA treatment, the R-Ras KO mice showed significantly lower levels of gene expression for IL-1α, IL-17A and IL-6 compared to WT (P = 0.0176, P = 0.032 and P = 0.0125 respectively, Fig. 5a). Gene expression of IL-17A and IL-6 in the R-Ras KO mice remained low at all of the time points tested. The level of IL-17A expression in the R-Ras KO mice remained low even at the 19-week time point whilst it was significantly elevated in the WT mice (P = 0.0189, Fig. 5a). The leukocyte count data showed the lack of a significant increase in skin macrophage infiltration or T-cells in the R-Ras KO mice at any time point, with neutrophil infiltration in the R-Ras KO mice only apparently elevated at 48 h after the second TPA treatment (Fig. 5b). In contrast, in the WT mice there was a clear increase in T-cell and macrophage numbers, which was significantly greater than in the R-Ras KO mice at the 48-h time point (P < 0.0001 for each, Fig. 5b). At the 19-week time point the WT mice had significantly more T-cells and especially more neutrophils than the R-Ras KO mice (P < 0.0001 for each, Fig. 5b). When comparing the timing of the inflammatory differences between the WT and R-Ras KO mice, it was noted that the lack of cytokine expression in the R-Ras KO mice was actually most evident preceding, not following, leukocyte infiltration (extravasation) (Fig. 5). The leukocyte count data in Fig. 5b is expressed as % of total nuclei rather than cells per mm2 as either format gave very similar results (Supplementary Figure S5a), but the former is more reliable in histological areas that are slightly damaged. A measure of total cells per mm2 in all groups revealed that there were no differences in total cell density per mm2 dermis between the WT and R-Ras KO mice, either before or after treatment (Supplementary Figure S5b).

Bottom Line: The R-ras gene encodes a small GTPase that is a member of the Ras family.Despite close sequence similarities, R-Ras is functionally distinct from the prototypic Ras proteins; no transformative activity and no activating mutations of R-Ras in human malignancies have been reported for it.The DMBA/TPA skin tumourigenesis-model is highly dependent upon inflammation, and we found a greatly attenuated skin inflammatory response to DMBA/TPA-treatment in the R-Ras KO mice in the context of leukocyte infiltration and proinflammatory cytokine expression.

View Article: PubMed Central - PubMed

Affiliation: School of Medicine, Department of Anatomy and Cell Biology, University of Tampere, Tampere, Finland.

ABSTRACT
The R-ras gene encodes a small GTPase that is a member of the Ras family. Despite close sequence similarities, R-Ras is functionally distinct from the prototypic Ras proteins; no transformative activity and no activating mutations of R-Ras in human malignancies have been reported for it. R-Ras activity appears inhibitory towards tumour proliferation and invasion, and to promote cellular quiescence. Contrary to this, using mice with a deletion of the R-ras gene, we found that R-Ras facilitates DMBA/TPA-induced skin tumour induction. The tumours appeared in wild-type (WT) mice on average 6 weeks earlier than in R-Ras knockout (R-Ras KO) mice. WT mice developed almost 6 times more tumours than R-Ras KO mice. Despite strong R-Ras protein expression in the dermal blood vessels, no R-Ras could be detected in the epidermis from where the tumours arose. The DMBA/TPA skin tumourigenesis-model is highly dependent upon inflammation, and we found a greatly attenuated skin inflammatory response to DMBA/TPA-treatment in the R-Ras KO mice in the context of leukocyte infiltration and proinflammatory cytokine expression. Thus, these data suggest that despite its characterised role in promoting cellular quiescence, R-Ras is pro-tumourigenic in the DMBA/TPA tumour model and important for the inflammatory response to DMBA/TPA treatment.

No MeSH data available.


Related in: MedlinePlus