guanosine-triphosphate and Papilloma

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

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