guanosine-triphosphate and Skin-Neoplasms

While several high-penetrance melanoma risk genes are known, variation in these genes fail to explain melanoma susceptibility in a large proportion of high-risk families. As part of a melanoma family sequencing study, including 435 families from Mediterranean populations we identified a novel NRAS variant (c.170A > C, p.D57A) in an Italian melanoma-prone family. This variant is absent in exomes in gnomAD, ESP, UKBiobank, and the 1000 Genomes Project, as well as in 11,273 Mediterranean individuals and 109 melanoma-prone families from the US and Australia. This variant occurs in the GTP-binding pocket of NRAS. Differently from other RAS activating alterations, NRAS D57A expression is unable to activate MAPK-pathway both constitutively and after stimulation but enhances EGF-induced PI3K-pathway signaling in serum starved conditions in vitro. Consistent with in vitro data demonstrating that NRAS D57A does not enrich GTP binding, molecular modeling suggests that the D57A substitution would be expected to impair Mg2 + binding and decrease nucleotide-binding and GTPase activity of NRAS. While we cannot firmly establish NRAS c.170A > C (p.D57A) as a melanoma susceptibility variant, further investigation of NRAS as a familial melanoma gene is warranted.

Topics: Cell Line, Tumor; Germ-Line Mutation; GTP Phosphohydrolases; Guanosine Triphosphate; Humans; Melanoma; Membrane Proteins; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-akt; Skin Neoplasms

The mouse skin model of carcinogenesis has been instrumental in our appreciation of the multistage nature of carcinogenesis. In this system, tumor promotion is a critical step in the generation of tumors and is usually achieved by treatment with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Although it is generally assumed that protein kinase C (PKC) is the sole receptor for TPA in this system, we sought to evaluate whether non-PKC pathways could also contribute to the effects of phorbol esters in skin. We documented expression of the high affinity non-PKC phorbol ester receptor and Ras activator RasGRP1 in mouse primary keratinocytes. Overexpression of RasGRP1 in keratinocytes increased the level of active GTP-loaded Ras. TPA treatment further elevated this Ras activation in a PKC-independent manner and induced the translocation and down-regulation of RasGRP1. Overexpression of RasGRP1 in keratinocytes also caused apoptosis. Finally, induction of keratinocyte differentiation by elevation of extracellular calcium suppressed expression of endogenous RasGRP1, whereas overexpression of RasGRP1 inhibited expression of the differentiation markers keratins 1 and 10 induced by high calcium in the medium. Taken together, our results demonstrate that RasGRP1 is an additional diacylglycerol/phorbol ester receptor in epidermal keratinocytes and suggest that activation of this novel receptor may contribute to some of the phorbol ester- and Ras-mediated effects in mouse epidermis.

Topics: Animals; Annexin A5; Apoptosis; Blotting, Western; Cell Differentiation; Cells, Cultured; DNA-Binding Proteins; Dose-Response Relationship, Drug; Down-Regulation; Epidermal Cells; Epidermis; Flow Cytometry; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate; Immunoblotting; Keratin-10; Keratinocytes; Keratins; Mice; Neoplasms, Experimental; Phorbol Esters; Precipitin Tests; Protein Kinase C; Protein Transport; Rats; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Skin Neoplasms; Tetradecanoylphorbol Acetate

Ultraviolet B (UVB) component of the sunlight is the major cause of nonmelanoma skin cancer (NMSC) in humans. UVB is absorbed directly by cellular DNA and produces lesions that may cause mutation(s) in target gene(s) ultimately leading to cancer. Early detection of these lesions, therefore, may help to identify individuals at a high risk to develop NMSC, and devise approaches for the prevention of this common malignancy. Employing mouse skin as a model, we applied a 32P postlabelling method to detect UVB-induced DNA lesions in the epidermis in nanomole quantities. Autoradiography maps showed that epidermal DNA from UVB exposed mice at 24 h contain up to five DNA lesions; the quantitation of these lesions showed that their formation increased in a UVB dose-dependent manner. Treatment of DNA samples with the bacteriophage DNA repair enzyme T4 endonuclease V confirmed that four of these lesions are pyrimidine dimers. While, some of these lesions were repaired 18 h after UVB irradiation, 30% of them persisted even 48 h post-irradiation. Application of a sunscreen containing ethylhexyl-p-methoxycinnamate or chemopreventive agent green tea polyphenols or silymarin to the skin of the mice prior to UVB exposure was found to prevent the formation of pyrimidine dimers.

Topics: Animals; DNA; Epidermis; Female; Guanosine Triphosphate; Mice; Mice, Hairless; Phosphorus Radioisotopes; Pyrimidine Dimers; Silymarin; Skin Neoplasms; Sunscreening Agents; Ultraviolet Rays

Epidermal papillomas were induced in CD-1 mice by a single topical application of 7,12-dimethylbenzanthracene (DMBA) followed by twice weekly applications of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in acetone. Control groups consisted of mice treated singly or chronically with acetone or TPA. TPA induced a rapid, yet transient 500- to 1000-fold increase in ornithine decarboxylase (ODC) activity which resulted in a 2- to 8.4-fold elevation of putrescine in both singly or chronically TPA-treated mouse epidermis 4-6 h after its application. After 24 h, levels of spermidine, but not spermine, were also elevated. The ODC and arginase activities in the 11 individual papillomas studied averaged 400- and 26-fold higher respectively than basal levels in epidermis. The activity of ODC in most papillomas, unlike ODC in epidermis, could be stimulated by guanosine 5'-triphosphate (GTP). Putrescine and spermidine levels in papillomas, especially those exhibiting highly GTP-stimulated ODC, were substantially higher compared to either normal or TPA-treated epidermis. Although epidermis contains a relatively high ornithine content, its level is even further elevated in papillomas, in some cases as much as 70-fold. The consequences of the constitutively elevated polyamine levels in papillomas caused by the loss of control over the normally tightly regulated polyamine biosynthetic pathway are not known, but could be important in regulating the balance between proliferation and differentiation in this self-renewing epithelial tissue.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Female; Guanosine Triphosphate; Mice; Mice, Inbred Strains; Ornithine; Ornithine Decarboxylase; Papilloma; Polyamines; Skin Neoplasms; Tetradecanoylphorbol Acetate

Previous studies have demonstrated the presence in mouse epidermal tumors of a structurally and functionally altered ornithine decarboxylase (ODC). In this report, the enzymatic properties of ODC from normal human skin and squamous cell carcinomas are examined. Some tumors contained a more heat stable ODC than the enzyme found in normal skin. GTP stimulated enzyme activity in four of seven tumor extracts tested but had no effect on normal skin ODC. Kinetic analyses indicated that GTP either lowered the apparent Km of tumor ODC for L-ornithine, increased the Vmax, or had both effects, depending on the tumor examined. Gel filtration chromatography of crude tumor extracts indicated the existence of multiple molecular weight forms of ODC, some of which can be activated by GTP and some of which are unaffected by GTP. Some tumors contain both a GTP-activatable and -nonactivatable form of the enzyme. Immunolocalization studies demonstrated the presence within squamous cell carcinomas of cells with a constitutively high level of immunoreactive ODC, a situation never observed in normal skin tissue. These results suggest that some human squamous cell carcinomas contain a functionally altered ODC that may be aberrantly regulated.

Topics: Carcinoma, Squamous Cell; Enzyme Activation; Guanosine Triphosphate; Humans; Kinetics; Ornithine Decarboxylase; Skin; Skin Neoplasms

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.

Topics: Animals; Catalysis; Chromatography; Enzyme Activation; Epidermis; Guanosine Triphosphate; Hot Temperature; Kinetics; Mice; Nucleotides; Ornithine Decarboxylase; Papilloma; Skin Neoplasms