Functional p53 protein in human papillomavirus-positive cancer cells.

There is accumulating evidence that the p53 protein contributes to tumor suppression by stimulating the transcription of specific cellular genes, such as the cell cycle control gene WAF1/ClP1. p53-mediated transcriptional activation is inhibited in cotransfection assays by overexpressed E6 protein from cancer-associated human papillomavirus (HPV) types, pointing at a possible molecular mechanism by which these viruses contribute to malignant cell transformation. Here we analysed the transcriptional transactivation function of endogenous p53 protein in a series of cervical cancer cell lines, which express the E6 gene from integrated viral sequences. Transient and stable transfection analyses employing p53-responsive reporter constructs indicated that HPV-positive cervical cancer cells contained transactivating p53 protein. Treatment of HPV-positive cells with genotoxic agents, such as mitomycin C, cisplatin, or u.v. irradiation, resulted in an increase of nuclear p53 protein levels and enhanced binding of p53 to a p53-recognition site. These effects were accompanied by an increase of WAF1/ClP1 mRNA levels. In several HPV-positive cell lines, these molecular events were linked to a cell cycle arrest in G1. In contrast, cancer cells containing mutant p53 genes did not contain transactivating endogenous p53 protein and lacked the p53-mediated response to DNA damaging agents. These results indicate that the tumorigenic phenotype of HPV-positive cancer cell lines does not necessarily correlate with a lack of basal or DNA damage induced p53 activities and that therefore the presence of high risk HPV sequences is not functionally equivalent to the loss of p53 function through somatic mutations of the p53 gene.

Characterization of skin reconstructed on a chitosan-cross-linked collagen-glycosaminoglycan matrix.

Reconstruction of skin requires both the dermal and epidermal equivalent of the skin. We have developed a reconstructed skin composed of two compartments: (1) a dermal equivalent comprising an acellular dermal substrate populated by foreskin fibroblasts and (2) an epidermis regenerated from normal human keratinocytes seeded onto the dermal equivalent. The dermal substrate contains type I and III collagen and glycosaminoglycans (GAGs) cross-linked by chitosan. Fibroblasts seeded into the porous structure of the dermal substrate provide a dermal equivalent suitable to support epidermal cells. Keratinocytes attach quickly, exhibit mitotic activity and form a continuous and stratified epidermis. After 2 weeks of culture, histological sections show a basal layer with cuboidal cells attached to the dermal equivalent and several suprabasal cell layers including the stratum corneum. Transmission electron microscopy revealed the cell membrane densification (hemidesmosomes) at the dermoepidermal junction; however, the lamina densa was found discontinuous at this stage. We noted the presence of lipid vesicles in spinous layer and keratohyalin granules in granular layer. The epidermal differentiation was complete terminal with the stratum corneum containing several layers of corneocytes filled with tonofilaments. Reconstructed skin, based on our chitosan-cross-linked collagen-GAG matrix is morphologically equivalent to normal human skin and should thus provide a useful tool for in vitro toxicological studies as well as a suitable wound covering for the treatment of patients with severe burns.