Myc-transformed epithelial cells down-regulate clusterin, which inhibits their growth in vitro and carcinogenesis in vivo.

Effective treatment of malignant carcinomas requires identification of proteins regulating epithelial cell proliferation. To this end, we compared gene expression profiles in murine colonocytes and their c-Myc-transformed counterparts, which possess enhanced proliferative potential. A surprisingly short list of deregulated genes included the cDNA for clusterin, an extracellular glycoprotein without a firmly established function. We had previously demonstrated that in organs such as skin, clusterin expression is restricted to differentiating but not proliferating cell layers, suggesting a possible negative role in cell division. Indeed, its transient overexpression in Myc-transduced colonocytes decreased cell accumulation. Furthermore, clusterin was down-regulated in rapidly dividing human keratinocytes infected with a Myc-encoding adenovirus. Its knockdown via antisense RNA in neoplastic epidermoid cells enhanced proliferation. Finally, recombinant human clusterin suppressed, in a dose-dependent manner, DNA replication in keratinocytes and other cells of epithelial origin. Thus, clusterin appears to be an inhibitor of epithelial cell proliferation in vitro. To determine whether it also affects neoplastic growth in vivo, we compared wild-type and clusterin-null mice with respect to their sensitivity to 7, 12-dimethylbenz(a)anthracene /12-Otetradecanoylphorbol-13-acetate (DMBA/TPA)-induced skin carcinogenesis. We observed that the mean number of papillomas/mouse was higher in clusterin-null animals. Moreover, these papillomas did not regress as readily as in wild-type mice and persisted beyond week 35. The rate of progression toward squamous cell carcinoma was not altered, although those developing in clusterin-null mice were on average better differentiated. These data suggest that clusterin not only suppresses epithelial cell proliferation in vitro but also interferes with the promotion stage of skin carcinogenesis.

TNF-α and IFN-γ are potential inducers of Fas-mediated keratinocyte apoptosis through activation of inducible nitric oxide synthase in toxic epidermal necrolysis.

Toxic epidermal necrolysis (TEN) is a severe immune-mediated adverse cutaneous drug eruption characterized by rapid and extensive epithelial cell death in the epidermis and mucosae. The molecular events leading to this often fatal condition are only partially understood, but evidence suggests a dual mechanism implicating a "drug"-specific immune response on one side and the onset of target cell death by proapoptotic molecules including FasL on the other side. Herein, we describe a potential molecular bridge between these two events that involves inducible nitric oxide synthase (iNOS), which is highly upregulated in the skin of TEN patients. We show that activated T cells secrete high amounts of tumor necrosis factor-α (TNF-α) and IFN-γ, and that both cytokines lead to increased expression and activity of keratinocyte iNOS. A similar observation has been made with drug-specific T lymphocytes from a TEN patient exposed to the culprit drug. The resulting increase in nitric oxide significantly upregulates keratinocyte FasL expression, resulting in Fas- and caspase-8-mediated keratinocyte cell death. Taken together, our data suggest that T-lymphocyte activation by drugs in TEN patients may indirectly lead to FasL-mediated keratinocyte apoptosis, via a molecular bridge involving TNF-α, IFN-γ, and iNOS.

Frequent loss of Fas expression and function in human lung tumours with overexpression of FasL in small cell lung carcinoma.

Fas (CD95) and its ligand FasL signal apoptosis and are involved in tissue homeostasis and the elimination of target cells by cytotoxic T cells. Corruption of this signalling pathway in tumour cells, for example by reduced Fas expression or increased FasL expression, can participate in tumour development and immune escape. The present study has analysed Fas/FasL expression and Fas death signalling function in vivo in lung tumour tissues [57 non-small cell lung carcinomas and 64 neuroendocrine lung tumours including small cell lung carcinoma (SCLC)] in comparison with normal lung tissue, and in vitro in neuroendocrine tumour cell lines in comparison with normal human bronchial epithelial cells. The Fas expression score was markedly decreased compared with normal lung tissue in 90% of the 121 lung tumours and was completely lost in 24%. The Fas staining pattern suggested cytoplasmic Fas expression in tumours, whereas membrane expression was observed in normal lung tissue. Loss of Fas at the cell surface was also shown in vitro by FACS analysis of neuroendocrine tumour cell lines and was concomitant with the resistance of tumour cells to FasL-mediated apoptosis according to in vitro cell viability. The lack of cell surface Fas expression in tumour cell lines resulted from the lack of intracellular Fas protein due to impaired Fas gene transcription. The FasL expression score was also decreased in most non-small cell lung carcinomas compared with normal bronchial cells, whereas 91% of SCLCs had higher expression than normal cells. FasL overexpression was related to advanced tumour stage as well as to a Fas/FasL ratio less than 1. It is concluded that a marked decrease in Fas expression may be part of lung tumourigenesis allowing tumour cells to escape from apoptosis. FasL overexpression in the context of Fas down-regulation in SCLC predicts the ability of SCLC cells to induce paracrine killing of Fas-expressing cytotoxic T cells. In lung tumours, Fas restoration may represent a key, although not unique, step in therapeutic strategies to reconstitute the ability of tumour cells to undergo apoptosis.

Intracellular localization of keratinocyte Fas ligand explains lack of cytolytic activity under physiological conditions.

Acquired Fas ligand (FasL)-mediated cytolytic activity of human keratinocytes causes the massive keratinocyte cell death that occurs during toxic epidermal necrolysis, a deadly adverse drug eruption. Under normal conditions keratinocyte apoptosis is a rare event in the epidermis although keratinocytes express the death receptor Fas and its ligand. Here we have investigated why this is so. We show that Fas, FasL, Fas-associated death domain, and caspase-8 mRNA are detectable in the epidermis, primary keratinocyte cultures, and keratinocyte cell line and that Fas protein is expressed in keratinocytes of all subcorneal layers of the epidermis, whereas FasL is only expressed in the basal and first suprabasal layers. Coexpression of Fas and FasL therefore occurs in basal and suprabasal keratinocytes. In vitro, keratinocytes are killed by recombinant FasL in a dose-dependent manner, but they are unable to kill Fas-sensitive target cells despite FasL expression. Analysis of keratinocyte culture supernatants and treatment of keratinocytes with metalloproteinase inhibitors excluded cell surface expression of FasL and rapid metalloproteinase-mediated cleavage of cell surface FasL. Fluorescence-activated cell sorter, confocal microscopical, and electron microscopical analysis revealed that keratinocyte FasL is localized intracellularly predominantly associated to intermediate filaments. These data suggest that the observed inability of keratinocyte FasL to induce apoptosis under physiological conditions is due to its cellular localization and also indicate that intermediate filaments may be involved in regulating the subcellular localization of FasL.