Abstract. In contrast to mouse epidermal cells, human skin keratinocytes are rather resistant to transformation in vitro. Immortalization has been achieved by SV40 but has resulted in cell lines with altered differentiation. We have established a spontaneously transformed human epithelial cell line from adult skin, which maintains full epidermal differentiation capacity. This HaCaT cell line is obviously immortal (>140 passages), has a transformed phenotype in vitro (clonogenic on plastic and in agar) but remains nontumorigenic. Despite the altered and unlimited growth potential, HaCaT cells, similar to normal keratinocytes, reform an orderly structured and differentiated epidermal tissue when transplanted onto nude mice. Differentiation-specific keratins (Nos. 1 and 10) and other markers (involucrin and filaggrin) are expressed and regularly located. Thus, HaCaT is the first permanent epithelial cell line from adult human skin that exhibits normal differentiation and provides a promising tool for studying regulation of keratinization in human cells. On karyotyping this line is aneuploid (initially hypodiploid) with unique stable marker chromosomes indicating monoclonal origin. The identity of the HaCaT line with the tissue of origin was proven by DNA fingerprinting using hypervariable minisatellite probes. This is the first demonstration that the DNA fingerprint pattern is unaffected by long-term cultivation, transformation, and multiple chromosomal alterations, thereby offering a unique possibility for unequivocal identification of human cell lines.

The restricted view of tumour progression as a multistep process defined by the accumulation of mutations in cancer cells has largely ignored the substantial contribution of the tumour microenvironment to malignancy. Even though the seed and soil hypothesis of Paget dates to 1889, it has been less than two decades since researchers have included the tumour microenvironment in their analyses of tumour progression. What have we recently learned from studying tumour-stroma interactions, and will this help to define new targets for therapy?

Acquisition of invasive/metastatic potential through protease expression is an essential event in tumor progression. High levels of components of the plasminogen activation system, including urokinase, but paradoxically also its inhibitor, plasminogen activator inhibitor 1 (PAI1), have been correlated with a poor prognosis for some cancers. We report here that deficient PAI1 expression in host mice prevented local invasion and tumor vascularization of transplanted malignant keratinocytes. When this PAI1 deficiency was circumvented by intravenous injection of a replication-defective adenoviral vector expressing human PAI1, invasion and associated angio-genesis were restored. This experimental evidence demonstrates that host-produced PAI is essential for cancer cell invasion and angiogenesis.Tumor cell invasion and metastatic processes require the coordinated and temporal regulation of a series of adhesive, proteolytic and migratory events 1 . The plasminogen activator (PA)-plasmin proteolytic system has been implicated in these processes. Urokinase-type (uPA) and tissue-type (tPA) plasminogen activators are serine proteases that catalyze the conversion of inactive plasminogen into plasmin, a broadly acting enzyme able to degrade a variety of extracellular matrix proteins and to activate metalloproteinases and growth factors 2,3 . Plasminogen and uPA bind to their specific receptors directing plasmin activity to the migrating tumor cell surface. The activities of PA are directly controlled by specific inhibitors, the PA inhibitors 1 and 2 (PAI1 and PAI2) (ref. 4).Many studies have focused on the role of uPA in cellular invasion and metastasis. Much of the data supporting the role of uPA in these events derives from in vitro and in vivo experiments demonstrating a correlation between uPA expression and cell invasion and metastasis as well as reduction of metastatic potential by using natural or synthetic serine protease inhibitors, neutralizing antibodies to uPA or antisense oligonucleotides 5,6 . PAI1 may also be directly involved in cancer progression. Both tumor cells and capillary endothelial cells express higher levels of PAI1 than other cell types [7][8][9] . Surprisingly, this inhibitor is necessary for optimal invasion of cultured lung cancer cells 10 , and an increasing number of clinical studies have demonstrated that high PAI1 levels indicate a poor prognosis for the survival of patients suffering from a variety of cancers [11][12][13] . However, as PAI1 is an acute-phase reactant 14 , it remains undetermined whether the increased PAI1 levels causally contribute to, or rather are the consequence of, the malignancy.Various observations indicate that the PA system may provide both surface-associated protease activity and an adhesion mechanism for cells through interaction with vitronectin. Deng et al. suggested that the balance between cell adhesion and cell detachment is governed by PAI1 (ref. 15). The PAI1-mediated release of cells attached to vitronectin seems to occur independently of the abili...

The plasminogen (Plg)/plasminogen activator (PA) system plays a key role in cancer progression, presumably via mediating extracellular matrix degradation and tumor cell migration. Consequently, urokinase-type PA (uPA)/plasmin antagonists are currently being developed for suppression of tumor growth and angiogenesis. Paradoxically, however, high levels of PA inhibitor 1 (PAI-1) are predictive of a poor prognosis for survival of patients with cancer. We demonstrated previously that PAI-1 promoted tumor angiogenesis, but by an unresolved mechanism. We anticipated that PAI-1 facilitated endothelial cell migration via its known interaction with vitronectin (VN) and integrins. However, using adenoviral gene transfer of PAI-1 mutants, we observed that PAI-1 promoted tumor angiogenesis, not by interacting with VN, but rather by inhibiting proteolytic activity, suggesting that excessive plasmin proteolysis prevents assembly of tumor vessels. Single deficiency of uPA, tissue-type PA (tPA), uPA receptor, or VN, as well as combined deficiencies of uPA and tPA did not impair tumor angiogenesis, whereas lack of Plg reduced it. Overall, these data indicate that plasmin proteolysis, even though essential, must be tightly controlled during tumor angiogenesis, probably to allow vessel stabilization and maturation. These data provide insights into the clinical paradox whereby PAI-1 promotes tumor progression and warrant against the uncontrolled use of uPA/plasmin antagonists as tumor angiogenesis inhibitors.

Although rodent cells have been immortalized following transfection with a mutant p53 gene, the role of p53 in the immortalization of human cells is unknown. Therefore, human epithelial cell lines were examined for p53 mutations in exons 4-9 which include the evolutionarily conserved regions. A spontaneously immortalized skin keratinocyte cell line, HaCat, and three ras-transfected clones, have a p53 mutational spectrum that is typical of ultraviolet light induced mutations. A normal finite lifespan cell strain (184) and two benzo[a]pyrene immortalized mammary epithelial cell lines derived from 184 (184A1 and 184B5) contain wild type p53 sequences in exons 4-9, although elevated levels of nuclear p53 indicate an alteration in the stability of the normally transient protein. Wild type p53 was found in human bronchial, esophageal and hepatic epithelial cells immortalized by SV40 T antigen gene and human renal epithelial cells immortalized by adenovirus 5. BEAS-2B, an SV40 T antigen immortalized bronchial epithelial cell line and two subclones, have a germline polymorphism at codon 47. Inactivation of p53 by mechanisms such as mutation or complexing with proteins of DNA tumor viruses appears to be important in the immortalization of human epithelial cells.

Interactions between mesenchymal and epithelial cells are responsible for organogenesis and tissue homeostasis. This mutual cross-talk involves cell surface proteins and soluble factors, which are mostly the result of regulated transcription. To elucidate dimer-specific functions of the AP-1 family of transcription factors, we reconstituted skin by combining primary human keratinocytes and mouse wild-type, c-jun(-/-), and junB(-/-) fibroblasts. We have discovered an antagonistic function of these AP-1 subunits in the fibroblast-mediated paracrine control of keratinocyte proliferation and differentiation, and traced this effect to the IL-1-dependent regulation of KGF and GM-CSF. These data suggest that the relative activation state of these AP-1 subunits in a non-cell-autonomous, transregulatory fashion directs regeneration of the epidermis and maintenance of tissue homeostasis in skin.

The immortal human keratinocyte line HaCaT is frequently used as a paradigm for skin keratinocytes in vitro because of its highly preserved differentiation capacity. HaCaT cells form a nearly regular epidermal architecture when transplanted onto subcutaneous tissue of athymic mice. In order to analyze further their differentiation capacity in vitro, HaCaT cells were studied in organotypic cocultures on top of collagen gels containing human dermal fibroblasts. Within 1 wk HaCaT cells formed a still dysplastic epithelium, the thickness of which correlated with the number of fibroblasts in the collagen gel. With further culture time of up to 3 wk a remarkably well structured and differentiated squamous epithelium developed. After 1 wk, keratins 10 and 16, involucrin, and transglutaminase I were expressed in suprabasal layers, whereas filaggrin, keratin 2e, and loricrin appeared after 2-3 wk. Within this time, a nearly complete basement membrane had formed including hemidesmosomes and anchoring fibrils. Epithelial cell proliferation became restricted to the basal layer after 2 and 3 wk. Using the TdT-mediated dUTP nick end labeling assay, fragmentation of DNA was detectable in nuclei of the parakeratotic stratum corneum. Ultrastructurally, many features of keratinization accumulated after 2 and 3 wk, though an orthokeratotic keratinization was not achieved, in contrast to HaCaT transplants. This differentiation deficiency - as compared with normal keratinocytes -- might be due to a lack of paracrine factors important for keratinocyte differentiation or to a reduced sensitivity of these cells. Nevertheless, this high degree of differentiation under organotypic conditions qualifies this cell line as an appropriate model for elucidation of the molecular mechanisms regulating keratinocyte growth and differentiation and for use in pharmacotoxicology.

Skin equivalents formed by keratinocytes cocultured with fibroblasts embedded in collagen lattices represent promising tools for mechanistic studies of skin physiology, for pharmacotoxicologic testing, and for the use as skin substitutes in wound treatment. Such cultures would be superior in defined media to avoid interference with components of serum or tissue extracts. Here we demonstrate that a defined medium (supplemented keratinocyte defined medium) supports epidermal morphogenesis in organotypic cocultures equally well as serum-containing medium (mixture of Ham's F12 and Dulbecco's modified Eagle's medium), as documented by hallmarks of the epidermal phenotype studied by immunofluorescence and electron microscopy. In both cases regularly structured, orthokeratinized epithelia evolved with similar kinetics. Morphology in mixture of Ham's F12 and Dulbecco's modified Eagle's medium was slightly hyperplastic, and keratins 1 and 10 synthesis less co-ordinated than in supplemented keratinocyte defined medium, but a consistently inverted sequence of expression of keratins 1 and 10 was found in either medium. The late differentiation markers filaggrin, involucrin, keratin 2e, and transglutaminase 1 corresponded in their typical distribution in upper suprabasal layers. Keratin 16 persisted under both conditions indicating the activated epidermal state. Keratinocyte proliferation was comparable in both media, whereas fibroblast multiplication and proliferation was delayed and reduced in supplemented keratinocyte defined medium. In both media, ultrastructural features of epidermal differentiation as well as reconstitution of a basement membrane occurred similarly. Immature lamellar bodies and cytoplasmatic vacuoles, however, indicated an impaired lipid metabolism in supplemented keratinocyte defined medium. Nevertheless, these defined organotypic cocultures provide a suitable basis for in vitro skin models to study molecular mechanisms of tissue homeostasis and for use in pharmacotoxicologic testing.