Friedrich Marks

Transgenic cyclooxygenase-2 overexpression sensitizes mouse skin for carcinogenesis

Genetic and pharmacological evidence suggests that overexpression of cyclooxygenase-2 (COX-2) is critical for epithelial carcinogenesis and provides a major target for cancer chemoprevention by nonsteroidal antiinflammatory drugs. Transgenic mouse lines with keratin 5 promoter-driven COX-2 overexpression in basal epidermal cells exhibit a preneoplastic skin phenotype. As shown here, this phenotype depends on the level of COX-2 expression and COX-2-mediated prostaglandin accumulation. The transgenics did not develop skin tumors spontaneously but did so after a single application of an initiating dose of the carcinogen 7,12-dimethylbenz[a]anthracene (DMBA). Long-term treatment with the tumor promoter phorbol 12-myristate 13-acetate, as required for tumorigenesis in wild-type mice, was not necessary for transgenics. The ratios of squamous cell carcinomas to papillomas and of sebaceous gland adenomas to papillomas plus squamous cell carcinomas were increased markedly in transgenic mice treated with DMBA alone compared with DMBA/phorbol 12-myristate 13-acetate-treated transgenic and wild-type mice. Thus, COX-2 overexpression, which leads to high levels of epidermal prostaglandin E2, prostaglandin F2α, and 15-deoxyΔ12,14-PGJ2, is insufficient for tumor induction but transforms epidermis into an “autopromoted” state, i.e., dramatically sensitizes the tissue for genotoxic carcinogens.

Abnormal differentiation of epidermis in transgenic mice constitutively expressing cyclooxygenase-2 in skin

In prostanoid biosynthesis, the first two steps are catalyzed by cyclooxygenases (COX). In mice and humans, deregulated expression of COX-2, but not of COX-1, is characteristic of epithelial tumors, including squamous cell carcinomas of skin. To explore the function of COX-2 in epidermis, a keratin 5 promoter was used to direct COX-2 expression to the basal cells of interfollicular epidermis and the pilosebaceous appendage of transgenic mouse skin. COX-2 overexpression in the expected locations, resulting in increased prostaglandin levels in epidermis and plasma, correlated with a pronounced skin phenotype. Heterozygous transgenic mice exhibited a reduced hair follicle density. Moreover, postnatally hair follicle morphogenesis and thinning of interfollicular dorsal epidermis were delayed. Adult transgenics showed a body-site-dependent sparse coat of greasy hair, the latter caused by sebaceous gland hyperplasia and increased epicutaneous sebum levels. In tail skin, hyperplasia of scale epidermis reflecting an increased number of viable and cornified cell layers was observed. Hyperplasia was a result of a disturbed program of epidermal differentiation rather than an increased proliferation rate, as reflected by the strong suppression of keratin 10, involucrin, and loricrin expression in suprabasal cells. Further pathological signs were loss of cell polarity, mainly of basal keratinocytes, epidermal invaginations into the dermis, and formation of horn perls. Invaginating hyperplastic lobes were surrounded by CD31-positive vessels. These results demonstrate a causal relationship between transgenic COX-2 expression in basal keratinocytes and epidermal hyperplasia as well as dysplastic features at discrete body sites.

Prostaglandin-H-synthase isozyme expression in normal and neoplastic human skin

Expression of prostaglandin‐H‐synthase (PGHS) isozymes was analyzed in 50 biopsies of normal human skin and of pre‐malignant and malignant skin lesions, by means of quantitative RT‐PCR, immunoprecipitation and Western blotting, as well as immunohistochemistry. Normal skin constitutively expressed PGHS‐1 in all cell layers of the epidermis, in endothelial cells of small blood vessels and in sweat‐gland epithelium. PGHS‐2 expression was very low and restricted to a few keratinocytes of the interfollicular and follicular epidermis. Steady‐state concentrations of PGHS‐1 and PGHS‐2 mRNA were similar in normal skin and in basal‐cell carcinomas, but PGHS‐1 mRNA was reduced and PGHS‐2 mRNA was elevated in actinic keratoses, squamous‐cell carcinomas and keratoacanthomas. PGHS‐1 protein was detected in all tumor biopsies, being occasionally increased in basal‐cell carcinomas. High amounts of PGHS‐2 protein were found in actinic keratoses, squamous‐cell carcinomas and keratoacanthomas, but not in basal‐cell carcinomas. Four malignant melanomas included in this study contained PGHS‐1 but no PGHS‐2 protein. Immunohistochemical analysis of the biopsies identified keratinocytes, in addition to cells of inflammatory infiltrates and of dendritic morphology, as the major PGHS‐expressing cell types. PGHS‐2‐specific signals were spread throughout the epidermal part of actinic keratoses and squamous‐cell carcinomas. These data suggest that constitutive up‐regulation of PGHS‐2 expression is a consistent pre‐malignant event in squamous‐cell cancer development in man, as it is in animal models of skin carcinogenesis. Thus, pre‐cancerous lesions such as actinic keratoses present a likely target for chemoprevention of skin cancer by selective PGHS‐2 inhibitors. Int. J. Cancer 82:648–656, 1999.

Metabolism and mechanism of action of oestrogens XII. Structure and mechanism of formation of water-soluble and protein-bound metabolites of oestrone in rat-liver microsomes in vitro and in vivo☆

Abstract 1. 1. During aerobic incubation of labeled oestrone or 2-hydroxyoestrone with rat-liver microsomes and NADPH, the steroid is bound to supernatant factors, e.g. glutathione and cysteine, and to microsomal proteins. Glutathione and protein compete for the same steroid metabolite. 2. 2. The binding reaction shows a remarkable specificity for sulfhydryl compounds; amino compounds are not bound. As far as the thiol is concerned, the binding reaction is nonspecific. A comparison of the properties of the metabolites, obtained by trapping the radioactivity with different thiols, demonstrates the formation of a thioether linkage. 3. 3. In carrier experiments with α-hydroxy-[4-14C]oestrone as substrate and with thioglycolic acid as trapping agent, mainly 2-hydroxy-4-carboxymethylmercapto-oestrone is formed. An o-quinone being excluded, these results suggest that the oestrogen metabolite bound by thiols is an o-semiquinone of 2-hydroxyoestrone. 4. 4. After injection of physiological doses of [6,7-3H2]oestrone, a long-lasting covalent protein binding can be demonstrated in rat liver in vivo. This reaction occurs especially in the microsomal fraction. 5. 5. The physiological significance of the binding reaction is discussed.

Protein-kinase-Cμ expression correlates with enhanced keratinocyte proliferation in normal and neoplastic mouse epidermis and in cell culture

In order to gain insight into the biological function of a PKC iso-enzyme, the protein kinase Cμ, we analyzed the expression pattern of this protein in mouse epidermis and keratinocytes in culture. Daily analysis of neonatal mouse epidermis immediately after birth showed a time-dependent reduction in the PKCμ content. Expression of the proliferating-cell nuclear antigen (PCNA), indicative of the proliferative state of cells, was reduced synchronously with PKCμ as the hyperplastic state of the neonatal tissue declined. In epidermal mouse keratinocytes, fractionated according to their maturation state, PKCμ expression was restricted to PCNA-positive basal-cell fractions. In primary cultures of those cells, growth arrest and induction of terminal differentiation by Ca2+ resulted in strongly reduced PKCμ expression, concomitantly with the loss of PCNA expression. Treatment of PMK-R1 keratinocytes with 100 nM of the mitogen 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in activation of PKCμ, reflected by translocation from the cytosolic to the particulate fraction and by shifts in electrophoretic mobility. DNA synthesis was significantly inhibited by the PKCμ inhibitor Goedecke 6976, while Goedecke 6983 did not inhibit PKCμ. Carcinomas generated according to the 2-stage carcinogenesis protocol in mouse skin consistently exhibited high levels of PKCμ. These data correlate PKCμ expression with the proliferative state of murine keratinocytes and point to a role of PKCμ in growth stimulation. A correlation between PKCμ expression and enhanced cell proliferation was also observed for NIH3T3 fibroblasts transfected with and over-expressing human PKCμ. Int. J. Cancer 80:98–103, 1999.

cDNA cloning of a 8-lipoxygenase and a novel epidermis-type lipoxygenase from phorbol ester-treated mouse skin

Using a combination of PCR cloning and conventional screening procedures, we isolated from phorbol ester-treated mouse epidermis two full length cDNA clones encoding novel lipoxygenases. One of the cDNAs turned out to be identical to the recently cloned 8-lipoxygenase [Jisaka et al., J. Biol. Chem. 272 (1997) 24 410-24 416], the open reading frame of the second one corresponded to a protein of 701 amino acids with a calculated molecular mass of 80.6 kDa. The amino acid sequence showed 50.8% identity to human 15-lipoxygenase 2, approximately 40% to 5-lipoxygenase and 35% to 12- and 15-lipoxygenases. A unique structural feature is the insertion of 31 amino acid residues in the amino-terminal part of the molecule. Based on these data, we conclude that this epidermis-derived cDNA encodes a novel lipoxygenase isoform termed provisionally epidermis-type lipoxygenase 2 (e-LOX 2).

Diversity of mouse lipoxygenases: Identification of a subfamily of epidermal isozymes exhibiting a differentiation-dependent mRNA expression pattern

By using reverse transcription-polymerase chain reaction technology (RT-PCR) and Northern blot analysis, the tissue-specific mRNA expression patterns of seven mouse lipoxygenases (LOX)—including 5S-, 8S-, three isoforms of 12S-, 12R-LOX, and a LOX of an as-of-yet unknown spacificity, epidermis-type LOX-3 (e-LOX-3)—were investigated in NMRI mice. Among the various tissues tested epidermis and forestomach were found to express the broadest spectrum of LOX. With the exception of 5S-and platelet-type 12S-LOX (p12S-LOX) the remaining LOx showed a preference to exclusive expression in stratifying epithelia of the mouse, in particular the integumental epidermis. The expression of the individual LOX in mouse epidermis was found to depend on the state of terminal differentiation of the keratinocytes. mRNA of epidermis-type 12S-LOX (e12S-LOX) was detected in all layers of neonatal and adult NMRI mouse skin, whereas experission of p12S-LOX, 12R-LOX, and e-LOX-3 was restricted to suprabasal epidermal layers of neonatal and adult mice. 8S-LOX mRNA showed a body-sitedependent expression in that it was detected in stratifying epithelia of footsole and forestomach but not in back skin epidermis. In the latter, 8S-LOX mRNA was strongly induced upon treatment with phorbol esters. With the exception of e12S-LOX and p12S-LOX, the isozymes that are preferentially expressed in stratifying epithelia are structurally related and may be grouped together into a distinct subgroup of epidermis-type LOX.

MURINE 12(R)-LIPOXYGENASE : FUNCTIONAL EXPRESSION, GENOMIC STRUCTURE AND CHROMOSOMAL LOCALIZATION

A cDNA, recently cloned (by Krieg et al. (1998)) from mouse skin, was shown to encode a 12(R)‐lipoxygenase. When expressed in HEK cells, the recombinant protein converted methyl arachidonate into the corresponding 12‐HETE ester which was shown to be the R‐enantiomer by chiral phase chromatography. Neither arachidonic acid nor linoleic acid were substrates for the recombinant protein. The structure of the 12(R)‐lipoxygenase gene is unique among all animal lipoxygenases in that it is divided into 15 exons and 14 introns spanning approximately 12.5 kb. By interspecific backcross analysis, the 12(R)‐lipoxygenase gene was localized to the central region of mouse chromosome 11.

Constitutive expression of 8-lipoxygenase in papillomas and clastogenic effects of lipoxygenase-derived arachidonic acid metabolites in keratinocytes.

The expression pattern, enzymatic activity, and products of 8‐lipoxygenase (LOX) were analyzed in normal and neoplastic skin of NMRI mice. While barely detectable in normal epidermis, 8‐LOX was transiently induced by 12‐O‐tetradecanoylphorbol‐13‐acetate and constitutively expressed in papillomas but not carcinomas obtained by the initiation‐promotion protocol of mouse skin carcinogenesis. The product profile and chirality of both the native and the recombinant protein produced the S enantiomers of 8‐hydroxy‐5Z,9E,11Z,14Z‐eicosatetraenoic acid (8‐HETE) and 9‐hydroxy‐10E,12Z‐octadecadienoic acid (9‐HODE) as the main arachidonic acid– and linoleic acid–derived metabolites. As compared with normal epidermis, papillomas exhibited 25‐ and 4‐fold elevated levels of 8‐HETE and 9‐HODE, respectively. However, the varying S to R ratios of 8‐HETE and the predominance of 9(R)‐HODE indicated that in addition to 8(S)‐LOX, other enzymes yet to be defined may be involved in 8‐HETE and 9‐HODE production. The massive accumulation of both 8‐HETE and 12‐hydroxy‐5Z,8Z,10E,14Z‐eicosatetraenoic acid (12‐HETE) point to a critical role of these LOX pathways in epidermal tumor development, in particular in the papilloma stage. Here we showed that 8‐ and 12‐hydroperoxyeicosatetraenoic acids and 8‐ and 12‐HETE induce chromosomal alterations in cycling primary basal keratinocytes. Mol. Carcinog. 24:108–117, 1999.

Murine epidermal lipoxygenase (Aloxe) encodes a 12-lipoxygenase isoform

Using a combination of conventional screening procedures and polymerase chain reaction cloning, we have isolated a cDNA encoding an epidermis‐type 12‐lipoxygenase (e12‐lipoxygenase) from mouse epidermis. The open reading frame corresponds to a protein of 662 amino acids and was found to be 99.8% identical to the ORF of an epidermal lipoxygenase gene Aloxe, described recently [Van Dijk et al. (1995) Biochim. Biophys. Acta 1259, 4–8]. When expressed in human embryonic kidney cells the recombinant protein could be shown to synthesize 12(S)‐HETE from arachidonic acid. By fluorescence in situ hybridization the e12‐lipoxygenase gene was localized to chromosome band 11 B1–B3.