Age-dependent skin tumorigenesis and transgene expression in the Tg.AC (v-Ha-ras) transgenic mouse.

Transgenic Tg.AC (v-Ha-ras ) mice develop skin tumors in response to specific carcinogens and tumor promoters. The Tg.AC mouse carries the coding sequence of v-Ha ras, linked to a zeta-globin promoter and an SV40 polyadenylation signal sequence. The transgene confers on these mice the property of genetically initiated skin. This study examines the age-dependent sensitivity of the incidence of skin papillomas in Tg.AC mice exposed to topically applied 12-O:-tetradecanoylphorbol-13-acetate (TPA) treatment, full thickness skin wounding or UV radiation. Skin tumor incidence and multiplicity were strongly age-dependent, increasing with increasing age of the animal when first treated at 5, 10, 21 or 32 weeks of age. Furthermore, the temporal induction of transgene expression in keratinocytes isolated from TPA-treated mouse skin was also influenced by the age of the mice. Transgene expression was seen as early as 14 days after the start of TPA treatment in mice that were 10-32 weeks of age, but was not detected in similarly treated 5-week old mice. When isolated keratinocytes were fractionated by density gradient centrifugation the highest transgene expression was found in the denser basal keratinocytes. Transgene expression could be detected in the denser keratinocyte fraction as early as 9 days from start of TPA treatment in 32-week old mice. Using flow cytometry, a positive correlation was observed between expression of the v-Ha-ras transgene and enriched expression of the cell surface protein beta1-integrin, a putative marker of epidermal stem cells. This result suggests that, in the Tg.AC mouse, an age-dependent sensitivity to tumor promotion and the correlated induction of transgene expression are related to changes in cellular development in the follicular compartment of the skin.

Endothelin-1 inactivating peptidase in the human kidney and urine.

OBJECTIVE: Recently, an apparently novel, specific endothelin-1 inactivating metalloendopeptidase (ET-1 peptidase) has been isolated from the rat kidney. In this study we attempted to determine whether the same or a similar peptidase is present in the human kidney, and whether the enzyme is excreted into the urine. The urinary ET-1 peptidase could serve as an indirect index of the renal endothelin system, both in physiology and pathophysiology. METHODS: Kidney specimens were obtained from part of nephrectomized kidneys unaffected by any neoplastic process from six adult patients. The enzyme was purified using differential centrifugation, detergent solubilization of the membrane proteins, ultrafiltration and nondenaturing gel electrophoresis. The enzyme activity assays were performed at pH 5.5 and 37 degrees C in the presence of increasing concentrations of unlabelled peptides and inhibitors using a fixed amount of [125I]ET-1 as substrate. The degradation extent was quantified with trichloroacetic acid precipitation and high performance liquid chromatography. The degrading activity of ET-1 was determined in urine samples from adult patients with hypertension, children with chronic renal failure and those with stable renal allograft RESULTS: ET-1 peptidase from the human kidney displays characteristics close to that of the rat ET-1 peptidase we have recently described (J. Hypertens 1994; 12:1155-1162). The enzyme, a membrane-bound metalloendopeptidase, exhibits low electro- phoretical mobility on nondenaturing gel (Rf 0.08); it is an apparently heterologous structure comprising three enzymatically inactive subunits, it has a pH optimum at 5.5, a nanomolar range affinity to the ET-1 (KM 180 nmol/l) that is hydrolysed to two main degradation products, and a 10-100-fold lower affinity to big ET-1 (KM 11.5 micromol/l), endothelin 11 21 fragment (KM 15.3 micromol/l), endothelin antagonist Trp-Leu-Asp-Ile-Ile-Trp (KM 3.1 micromol/I), gastrin (KM 2.2 micromol/l) and cholecystokinin (KM 4.0 micromol/l). Substance P, neuropeptide Y, atrial natriuretic peptide, bradykinin, angiotensin II and enkephalin were poor substrates for the enzyme. The most powerful inhibitors of the ET-1 peptidase included thiorphan (IC50 0.28 nmol/l), phosphoramidon (IC50 0.55 nmol/l), phenanthroline (IC50 11.5 micromol/l), cyclosporin (IC50 400 micromol/l), phosphate (IC50 1.2 mmol/l), citrate (IC50 0.6 mmol/l) and aniline naphthalene sulphonic acid (IC50 0.25 mmol/l). Our data suggest that three ET-1 degrading peptidases with optimal activity at pH 4.5, 5.5 and 7.0, respectively, are excreted into the urine. The enzyme with a pH optimum 4.5 is of lysosomal origin whereas the two other enzymes correspond by their pH optima to the renal ET-1 peptidase and neutral endopeptidase. We have found statistically significant increases (P < 0.001) in the activity of both lysosomal and ET-1 peptidase in the urine in patients with hypertension and in children with chronic renal failure compared with healthy subjects or children with stable renal allograft CONCLUSIONS: Human kidney contains an acidic, highly specific endothelin-1 inactivating metalloendopeptidase that may have a key role in the regulation of concentrations of renal and circulating endothelins. The enzyme is excreted into the urine where its activity seems to be increased in patients with hypertension and chronic renal failure; it may potentially serve as an indirect index of the renal endothelin system.

TGF alpha is dispensable for skin tumorigenesis in Tg.AC mice.

Alterations in growth factor signaling pathways frequently accompany the development and maintenance of epithelial neoplasia. Transforming growth factor alpha (TGF alpha) and its epidermal growth factor receptor have been thought to play an especially important role in epithelial neoplasia. In this study, mice were derived genetically deficient (null) in functional TGF alpha expression and carrying the Tg.AC/v-Ha-ras transgene. The goals were to determine if (a) papillomagenesis was dependent on TGF alpha and (b) progression to malignancy was dependent on TGF alpha expression. Groups of male and female mice heterozygous or homozygous for the TGF alpha null allele and hemizygous for the Tg.AC transgene were treated twice weekly for 10 or 15 wk with doses of 12-O-tetradecanoylphorbol-13-acetate (TPA) known to produce papillomas in Tg.AC mice. Papillomas were readily induced in both male and female TGF alpha null mice. Malignant progression of papillomas was observed in all TGF alpha null treatment groups. Additionally, we examined the response of TGF alpha null mice to full thickness dorsal wounds, a stimulus known to promote papillomagenesis in Tg.AC mice. As in the TPA study, papillomas were induced in both male and female TGF alpha null mice. These studies indicate that TGF alpha is not required for the induction and maintenance of papillomas nor is it essential for the malignant conversion of papillomas in Tg.AC mice.

Kinetics of wound-induced v-Ha-ras transgene expression and papilloma development in transgenic Tg.AC mice.

The Tg.AC transgenic mouse, which harbors an activated v-Ha-ras coding region that is fused to an embryonic zeta globin transcriptional control region and a 3' simian virus 40 polyadenylation sequence, rapidly develops epidermal papillomas in response to topical application of chemical carcinogens or tumor promoters or to full-thickness wounding of the dorsal skin. In this report, we investigated the localization and temporal induction of v-Ha-ras transgene expression after full-thickness wounding of Tg.AC mouse skin. Surgically inflicted full-thickness incisions 3 cm long yielded four to six papillomas per Tg.AC mouse by 5 wk after wounding. Similar wounding of the FVB/N isogenic host strain did not produce tumors, which implicates a causal role for the v-Ha-ras transgene. Reverse transcription-polymerase chain reaction assays detected the v-Ha-ras transgene transcript in total RNA samples isolated from wound-associated tissue 3 and 4 wk after wounding. Tissues 1-2 wk after wounding and all non-wound-associated tissues were negative for transgene expression. In situ hybridization experiments using transgene-specific 35S-labeled antisense RNA probes localized transgene expression to the basal epidermal cells in wound-induced papillomas. Adjacent normal and hyperplastic skin tissues were negative for transgene expression by this assay. This work supports the hypothesis that the wound repair response leads to the transcriptional activation and continued expression of the v-Ha-ras transgene in specific cells in the skin, which alters normal epithelial differentiation and ultimately results in neoplastic growth.

Odontogenic tumours in the v-Ha-ras (TG.AC) transgenic mouse.

A line of homozygous transgenic mice (TG.AC) carrying a v-Ha-ras gene fused to the promoter of the zeta globin gene produces a variety of mesenchymal and epithelial neoplasms including odontogenic tumours. The 1-year incidence of odontogenic tumour formation in these mice was approx. 35%. Tumours formed more often in the mandible than maxilla. The various types of tumours frequently presented with: (1) primarily mesenchymal cells in a dense fibrous-like matrix, or (2) loose stroma surrounded by anastomosing cords of epithelial cells that exhibited squamous differentiation, or (3) odontomas forming mineralized tooth structures by well-differentiated odontoblasts and ameloblasts. Some tumours had areas with all three of these characteristics. Mineralized dentine and enamel in the odontomas were morphologically similar to those of normal murine teeth. Odontogenic tumours expressed the v-Ha-ras transgene that was primarily localized to the mesenchymal cells. Proliferating-cell nuclear antigen immunohistochemistry showed that the mesenchymal cells adjacent to the epithelial cords not only expressed the ras transgene but were also actively proliferating. The TG.AC mouse provides an excellent model for the study of odontogenic tumours and tooth development.