David A. Greenhalgh

Mutations in the rod domains of keratins 1 and 10 in epidermolytic hyperkeratosis.

Epidermolytic hyperkeratosis is a hereditary skin disorder characterized by blistering and a marked thickening of the stratum corneum. In one family, affected individuals exhibited a mutation in the highly conserved carboxyl terminal of the rod domain of keratin 1. In two other families, affected individuals had mutations in the highly conserved amino terminal of the rod domain of keratin 10. Structural analysis of these mutations predicts that heterodimer formation would be unaffected, although filament assembly and elongation would be severely compromised. These data imply that an intact keratin intermediate filament network is required for the maintenance of both cellular and tissue integrity.

Expression of a p53 mutant in the epidermis of transgenic mice accelerates chemical carcinogenesis.

To develop an in vivo model for studying the role of the p53 tumor suppressor in skin carcinogenesis, a murine p53172H mutant (equivalent to human p53175H) was expressed in the epidermis of transgenic mice, utilizing a targeting vector based on the human keratin 1 gene (HK1.p53m). HK1.p53m mice developed normally and did not exhibit an obvious epidermal phenotype or develop spontaneous tumors. However, these mice demonstrated an increased susceptibility to a two-stage chemical carcinogenesis protocol, with the rate of formation and number of papillomas being dramatically increased as compared to non-transgenic controls. The majority of papillomas in control mice regressed after termination of 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment, whereas p53m papillomas progressed to carcinomas and metastases. In addition, more advanced malignancy, i.e., undifferentiated spindle cell carcinomas, were exclusively observed in p53m mice. Increased bromodeoxyuridine (BrdU) labeling, accompanied by decreased expression of p21, was observed in HK1.p53m papillomas. In situ examination of centrosomes in HK1.p53m papillomas also revealed marked abnormalities, with 75% of the cells containing ⩾3 centrosomes/cell, whereas centrosome numbers in papillomas from control animals remained normal. These data suggest that the accelerated tumorigenesis observed in chemically-treated p53m mice is most likely due to increased genomic instability resulting from an inhibition of G1 arrest and abnormal amplification of centrosomes.

Human keratin-1. bcl -2 transgenic mice aberrantly express keratin 6, exhibit reduced sensitivity to keratinocyte cell death induction, and are susceptible to skin tumor formation

Nonmelanoma skin cancers (NMSC) are among the most common malignancies in the world. Typically, these neoplasms grow slowly and are comparatively indolent in their clinical behavior. The most frequent molecular alterations implicated in the pathogenesis of these neoplasms involve genes known to be regulators of cell death including p53, Ha-ras and bcl-2. In order to evaluate the significance cell death deregulation during skin carcinogenesis, we generated a transgenic mouse model (HK1.bcl-2) using the human keratin 1 promoter to target the expression of a human bcl-2 minigene to the epidermis. Transgenic HK1.bcl-2 protein was expressed at high levels specifically in the epidermis extending from the stratum basale through the stratum granulosum. The epidermis of HK1.bcl-2 mice exhibited multifocal hyperplasia without associated hyperkeratosis and aberrant expression of keratin 6. The rate of proliferation was similar in HK1.bcl-2 and control epidermis although suprabasal BrdUrd incorporating cells were present only in HK1.bcl-2 skin. Keratinocytes from the HK1.bcl-2 mice were significantly more resistant to cell death induction by U.V.-B, DMBA, and TPA, compared to control keratinocytes. Furthermore, papillomas developed at a significantly greater frequency and shorter latency in the HK1.bcl-2 mice compared to control littermates following initiation with DMBA and promotion with TPA. Together these results support a role for bcl-2 in the pathogenesis of NMSC.

Analysis of centrosome abnormalities and angiogenesis in epidermal-targeted p53172H mutant and p53-knockout mice after chemical carcinogenesis: evidence for a gain of function.

We previously developed a transgenic mouse model that expresses in the epidermis a murine p53172R→H mutant (p53m) under the control of a human keratin‐1–based vector (HK1.p53m). In contrast to mice with wild‐type p53 and p53‐knockout mice, HK1.p53m mice exhibit increased susceptibility to chemical carcinogenesis, with greatly accelerated benign papilloma formation, malignant conversion, and metastasis. In the study presented here, we examined the expression pattern of several differentiation markers and observed that p53m tumors exhibited a less differentiated phenotype than tumors elicited in non‐transgenic mice. Metastasis in p53m tumors was also associated with a poorly differentiated phenotype. To determine whether genomic instability was associated with a putative gain‐of‐function role for this p53m, in situ examination of centrosomes was performed in HK1.p53m and equivalent p53‐null papillomas. In contrast to HK1.p53m papillomas, which had centrosome abnormalities at high frequencies (75% of cells contained more than three centrosomes/cell), p53‐null tumors exhibited few abnormal centrosomes (4% of cells contained more than three centrosomes/cell). To determine whether angiogenesis played a role in the rapid progression of p53m tumors, the expression of vascular endothelial growth factor, a promoter of angiogenesis, and thrombospondin‐1, an inhibitor of angiogenesis, was examined in tumors derived from either p53m or p53‐knockout mice. Regardless of their p53 status (wild type, p53m, p53–/–), all of the papillomas exhibited similar levels of vascular endothelial growth factor expression and decreased expression of thrombospondin‐1 as did normal epidermis. In addition, tumors from different p53 genotypes showed a similar density of blood vessels. Because p53 status did not appear to play an overt role in angiogenesis, these data suggest that p53m accelerates tumorigenesis primarily by exerting a gain of function associated with genomic instability. Mol. Carcinog. 23:185–192, 1998.

Dissecting Molecular Carcinogenesis: Development Of Transgenic Mouse Models By Epidermal Gene Targeting

Publisher Summary This chapter focuses on the attempts to target gene expression to the epidermis and to develop models that will be relevant to both the skin carcinogenesis in particular and the epithelial carcinogenesis in general. It highlights the power of gene targeting to study carcinogenesis in vivo. An epidermal targeting vector have been developed and transgenic mice is established that express Ha- ras, fos, transforming growth factor α (TGFa), and the E6/E7 transforming genes of human papillomavirus type 18 (HPV-18) exclusively in the epidermis. Although the individual genes have distinct phenotypic characteristics, the stability of the preneoplastic and premalignant phenotypes produced by each line over extended time periods is consistent. This stability indicates not only the necessity of secondary events for progression, but also demonstrates that this transgenic mouse model appears to be ideally qualified to assess the nature of these events. In vivo co-operation experiments are described that assess the consequences of acquisition of an additional genetic event. The chapter evaluates potential application of these human keratin K1 gene (HK1) transgenic mice as environmental carcinogen/promoter test systems.

Adenovirus-Mediated Ex Vivo Immunogene and in Vivo Combination Gene Therapy Strategies Induce a Systemic Anti-Tumor Immune Defense in the Mouse B16 Melanoma Model

Most of the gene therapy studies in melanoma which are ongoing or have been performed in the recent past (clinical phase I/II trials) rely on a retroviral and an in vitro/ex vivo gene transfer [reviewed in 1,2]. As an alternative, we studied a recombinant, replication-deficient adenovirus (adv, serotype 5) system for its properties to transduce murine and human melanoma cells under in vivo and in vitro (ex vivo) gene transfer conditions in preclinical models [3–5]. Adv has already been translated to the clinical field by several current trials investigating the use of adv vectors for the treatment of cystic fibrosis [6]. In contrast to retroviruses, adv can be enriched to high titers because of its particular physico-chemical stability [7,8]. Moreover, adenoviral cell transduction does not depend upon division of the host cells, and adv sequences regularly do not integrate into the host genome, resulting in the added safety feature of little significant potential for insertional mutagenesis [7,8]. With regard to retroviral vectors, much concern arose when 3 of 10 primates developed T-cell lymphomas after immunosuppressive body irradiation and subsequent transfusion of autologous bone marrow cells, which had been genetically modified by a retroviral vector in vitro, and were also contaminated with replication competent retrovirus [9]. Wild-type adv seems to be more safe as proven by broad vaccination campaigns with oral administration of enteric-coated capsules containing live, unattenuated adv resulting only rarely in side effects [10,11]. Furthermore, the serotypes 2 and 5 of adv which are currently used as non-replicating vectors are not tumorigenic in rodents, and replicating wild types cause only mild respiratory infections in humans [10].