Absence of Host Plasminogen Activator Inhibitor 1 Prevents Cancer Invasion and Vascularization

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 angiogenesis were restored. This experimental evidence demonstrates that host-produced PAI is essential for cancer cell invasion and angiogenesis

Sensitization of human keratinocytes to killing by parvovirus H-1 takes place during their malignant transformation but does not require them to be tumorigenic

To investigate the antineoplastic activity of parvoviruses, proliferating normal human epidermal cells and a series of established keratinocyte cell lines derived from squamous cell carcinomas or transformed in vitro, were compared for the outcome of H-1 virus infection. All established keratinocyte cell lines were more sensitive to killing by H-1 virus than normal epidermal cells, although to varying extents. Using a step-wise procedure for malignant transformation in vitro, we found that sensitization of transformed epidermal cells to H-1 virus can be dissociated from the acquisition of a tumorigenic phenotype. Thus, spontaneously- or SV40-immortalized human keratinocytes were moderately and highly sensitive to H-1 virus, respectively, and could be made tumorigenic by Harvey-ras oncogene transfection without a major change in their susceptibility to the virus. The capacity of human keratinocytes for replicating and expressing H-1 virus DNA appears to be a revealer of cellular alterations that take place in at least some pathways to malignant transformation but that may be insufficient to confer a tumorigenic potential.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Cutaneous basement membrane formation in organotypic culture

The cutaneous basement membrane (BM) consists mainly of polymeric collagen-IV and laminin-10 and associated mono-/oligomeric laminin-5, nidogen, and perlecan. Since BM-defects in transgenic or knockout mice are mostly lethal at early developmental stages, we have studied the role of nidogen specifically in 3D-cocultures of human keratinocytes (HK) and fibroblasts (human/mouse, HF/MF) by either blocking interactions or implementing molecular deficiencies. HK or HaCaT cells were grown on collagen gels harboring HF or MF from normal or ko-mice. Nidogen-laminin interaction was blocked by the laminin-fragment (gamma-1-III3-5, L-gamma-f) binding nidogen. BM-formation was surveyed by immunofluorescence (IF), regular (EM), immuno-electron microscopy (IEM), and Western blots of protein extracts of separated epithelial and 'dermal' tissue. In 3D-cocultures of HK and HF L-gamma-f blocked mainly deposition of nidogen, laminin-10, and perlecan. Whereas the hemidesmosomal/BM components laminin-5, BP180, and integrin alpha6beta4 were still detectable (IF), by EM and IEM any BM-structures or hemidesmosomes (insertion of keratins) were absent. The fibroblast-made nidogen was eliminated by employing MF from nidogen-1/-2 ko-mice. In 3D-cocultures with HaCaT cells nidogen1/2 (--/++)-MF abolished nidogen-1 staining, but (--/+-)-MF reduced additionally nidogen-2, collagen-IV, and laminin-10. Absence of nidogens (--/--) further abolished collagen-IV and laminin-5; integrins such as alpha6beta4 appear normal (IIF). BM-formation could be reinstalled with recombinant nidogen-1 or -2. BM-perlecan, for comparison, is apparently synthesized also by keratinocytes. Thus, deficiency in either cell type did not affect BM-formation, demonstrated by growing perlecan (-/-)-MF or HaCaT antisense-perlecan cells with normal keratinocytes or fibroblasts, respectively. Accordingly, BM-components are efficiently recruited for ultrastructural assembly in this skin model

Control of basement membrane formation in skin-organotypic 3d-coculture

Basement membrane (BM) formation was functionally dissected in 3d-cocultures of human keratinocytes (HK) and fibroblasts (human/mouse, HF/MFf) by either blocking interactions or implementing molecular deficiencies. This was supposed to complement knockout mouse studies, where loss or functional defects of collagen-IV, laminins, nidogen, or perlecan are causing embryonic or neonatal death. HK or HaCaT cells were grown on collagen gels harboring hf or mf from normal or ko-mice. To block nidogen-binding to laminin-10 the corresponding laminin-fragment (gamma1-iii3-5, L-gamma-f) was applied. BM-formation was surveyed by immunofluorescence (IF), regular (EM) and immuno-electron microscopy (IEM). In 3d-cocultures of HK and HF L-gamma-f blocked deposition of nidogen, laminin-10, and perlecan, while collagen-IV appeared normal. Although the hemidesmosome components laminin-5, BP180, and integrin alpha6beta4 were only mildly affected, EM and IEM revealed complete absence of BM, hemidesmosomes, and basal insertion of keratin filaments. To eliminate nidogen, made by fibroblasts, MF from nidogen1/nidogen2 ko-mice or crossbreds were employed. In 3d-cocultures with HaCaT cells nidogen1/2 (??/++)-MF abolished nidogen1-staining, but (??/+?)-mf reduced also largely nidogen2, collagen-IV, and drastically laminin-10. Total absence of nidogen (??/??) also deleted collagen-IV & laminin-5, integrins e.g. alpha6beta4 appearing still normal (IF). BM-formation could be entirely rescued by applying recombinant nidogens. In skin, perlecan can be apparently synthesized by both keratinocytes & fibroblasts. Accordingly, deficiency in either cell type did not affect BM-formation, demonstrated by combining either perlecan (?/?)-mf or HaCaT anti-sense-perlecan cells with respective normal partner cells. Thus, in this skin model BM-components are efficiently transported to their actual assembly site