Regulation of ornithine decarboxylase gene expression in mouse epidermis and epidermal tumors during two-stage tumorigenesis.

Topical treatment of mouse skin with the potent tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) results in an array of biochemical alterations, one of the earliest being a more than 200-fold transient induction of epidermal ornithine decarboxylase (ODC) activity. There is an excellent correlation between the induction of epidermal ODC activity and changes in the level of immunoreactive ODC protein following a single TPA treatment to skin. Both ODC activity and protein levels peak at 4.5 h after TPA treatment and rapidly fall to basal levels by 24 h. Cycloheximide treatment of mice in which ODC had been previously induced by TPA indicated a similar rapid turnover of both ODC catalytic activity and protein levels. Northern blot analysis of polyadenylated RNA isolated from mouse epidermis after a single TPA treatment revealed the stimulation of one species of ODC mRNA of 2.0 kilobases with a maximum at 3.5 h declining by 16 h. The same-sized species of ODC mRNA was detected 4.5 h after multiple biweekly treatments with TPA as well as in mouse papillomas and carcinomas not treated with TPA for at least 1 week. Southern blot analysis of EcoRI or BamHI digests of DNA derived from mouse liver, papillomas, or carcinomas revealed no ODC gene amplification or rearrangement during neoplastic progression. These observations indicate that the induction of epidermal ODC activity following TPA treatment results in a transient increase in the steady state levels of ODC mRNA and in the rate of synthesis of ODC protein, in contrast to epidermal tumors where the levels of ODC mRNA and protein are constitutively elevated.

Ornithine decarboxylase from mouse epidermis and epidermal papillomas: differences in enzymatic properties and structure.

The properties of ornithine decarboxylase (OrnDCase) from mouse epidermis and benign epidermal tumors (papillomas) induced by the initiation-promotion protocol were compared. When crude extracts from each tissue were incubated at 55 degrees C, epidermal OrnDCase was rapidly inactivated, but the papilloma OrnDCase was more heat stable. Each of five individual papilloma extracts contained OrnDCase activity that was considerably more resistant to heat inactivation than was epidermal OrnDCase. Mixing of a papilloma and epidermal extract produced an intermediate heat-inactivation profile, suggesting that the differences in OrnDCase heat stability are not due to non-OrnDCase components of the extracts. Kinetic analyses indicated that the papilloma OrnDCase has an altered affinity for its substrate, L-ornithine, compared to epidermal OrnDCase. The apparent Km for L-ornithine for the epidermal enzyme was 0.07 mM while the Km values for the individual papilloma OrnDCases clustered around two higher values, 0.3 mM and 1.0 mM. The papilloma OrnDCases, but not epidermal OrnDCase, were activated by GTP and to a lesser extent by CTP. Immunoblot analysis showed the existence of multiple forms of OrnDCase in both epidermis and papilloma that differed in isoelectric point but not subunit molecular weight. None of the species of OrnDCase present in the epidermal extract coincided with the species present in papilloma. These results suggest that one consequence of neoplastic transformation in this in vivo system is the presence of an OrnDCase protein in benign tumors that differs structurally and functionally from the OrnDCase present in normal epidermis. The possible mechanisms responsible for these results and their significance for neoplastic development in this tissue are discussed.

Induction of ornithine decarboxylase by 12-O-tetradecanoylphorbol 13-acetate in hamster fibroblasts. Relationship between levels of enzyme activity, immunoreactive protein, and RNA during the induction process.

Phorbol ester tumor promoters and growth factors rapidly stimulate ornithine decarboxylase activity in the transformed hamster fibroblast line HE68BP. We report here a close correspondence between the time courses and magnitudes of induction of ornithine decarboxylase activity and immunoreactive ornithine decarboxylase protein following treatment of HE68BP cells with 12-O-tetradecanoylphorbol 13-acetate (TPA) and/or refeeding with fresh medium. Cycloheximide addition to induced cells caused a rapid fall in the levels of both ornithine decarboxylase activity and ornithine decarboxylase protein. Northern blot analysis of RNA isolated from HE68BP cells indicated that treatment with TPA and fresh medium increased the amount of two species of mRNA of lengths 2.4 and 2.1 kilobase. This increased accumulation of ornithine decarboxylase mRNA corresponded temporally to that observed at the protein level, with a 15-fold maximal induction 7 h after treatment followed by a rapid decline in hybridizable RNA. These data indicate that stimulation of ornithine decarboxylase activity by TPA or refeeding involves changes in levels of ornithine decarboxylase mRNA as well as changes in the rate of synthesis of ornithine decarboxylase protein.

Glycoprotein D protects mice against lethal challenge with herpes simplex virus types 1 and 2.

Glycoprotein D is a virion envelope component of herpes simplex virus types 1 and 2. Sets of mice were immunized with purified gD-1 or gD-2 and were challenged with a lethal dose of herpes simple virus, either type 1 or type 2. All or virtually all of the immunized mice survived challenge with either agent, whereas challenge of sham-immunized mice was almost always fatal. Serum samples taken before challenge contained gD-specific antibodies which had 50% neutralization titers ranging from 1:16 to 1:512 against homologous and heterologous virus types. We conclude that either gD-1 or gD-2 is a potential candidate for a subunit vaccine against herpetic infections.

Effects of UV irradiation on the fate of 5-bromodeoxyuridine-substituted bacteriophage T4 DNA.

We have carried out a series of experiments designed to characterize the impact of UV irradiation (260 nm) on 5-bromodeoxyuridine-labeled (heavy) T4 bacteriophage, both before and after infection of Escherichia coli. In many respects, these effects differ greatly from those previously described for non-density-labeled (light) phage. Moreover, our results have led us to propose a model for a novel mechanism of host-mediated repair synthesis, in which excision of UV-damaged areas is followed by initiation of replication, strand displacement, and a considerable amount of DNA replication. UV irradiation of 5-bromodeoxyuridine-labeled phage results in single-stranded breaks in a linear, dose-dependent manner (1.3 to 1.5 breaks per genomic strand per lethal hit). This damage does not interfere with injection of the phage genome, but some of the UV-irradiated heavy phage DNA undergoes additional intracellular breakdown (also dose dependent). However, a minority (25%) of the injected parental DNA is protected, maintaining its preinjection size. This protected moiety is associated with a replicative complex of DNA and proteins, and is more efficiently replicated than is the parental DNA not so associated. Most of the progeny DNA is also found with the replicative complex. The 5-bromodeoxyuridine of heavy phage DNA is debrominated by UV irradiation, resulting in uracil which is removed by host uracil glycosylase. Unlike the simple gap-filling repair synthesis after infection with UV-irradiated light phage, the repair replication of UV-irradiated heavy phage is extensive as determined by density shift of the parental label in CsC1 gradients. The newly synthesized segments are covalently attached to the parental fragments. The repair replication takes place even in the presence of chloramphenicol, a protein synthesis inhibitor, suggesting it is host mediated. Furthermore, the extent of the repair replication is greater at higher doses of UV irradiation applied to the heavy phage. This abundant synthesis results ultimately in dispersion of the parental sequences as short stretches in the midst of long segments of newly synthesized progeny DNA. Together, the extensive replication and the resulting distribution pattern of parental sequences, without significant solubilization of parental label, are most consistent with a model of repair synthesis in which the leading strand displaces, rather than ligates to, the encountered 5' end.