Notch receptors play a key role in several cellular processes including differentiation, proliferation, and apoptosis. This study investigated whether the activation of Notch signaling would affect the maturation of dendritic cells (DCs). Direct stimulation of Notch signaling in DCs with a peptide ligand induced DC maturation, similar to LPS: DCs up-regulated maturation markers, produced IL-12, lost endocytosis capacity, and became able to activate allogeneic T cells. Furthermore, coculture of DCs with cells expressing Notch ligand Jagged-1 induced up-regulation of maturation markers, IL-12 production, T cell proliferative responses, and IFN-γ production. Our data suggest that activation of Notch by Jagged-1 plays an important role in maturation of human DCs. Additionally, they reveal a novel role for Notch signaling in cell maturation events distal to the cell fate decision fork. These data may have important medical implications, since they provide new reagents to induce DC activity, which may be beneficial as adjuvants in situations where an immune response needs to be elicited, such as tumor immunotherapy.

Apoptosis is generally regarded as a critical regulatory event in the development of malignancies in several different organ systems (Thompson, 1995). Initially, oncologists focused on alterations in rates of proliferation and cell cycle kinetics, but more recently an emphasis on apoptosis has dominated the fight against cancer (Evan and Vousden, 2001). As approximately 1,000,000 individuals in the U.S.A. develop skin cancer each year, it is important to elucidate the molecular mechanisms that govern cell survival and cell death in the epidermis (Miller and Weinstock, 1994). Moreover, given that most skin cancers occur on sun-exposed skin, the pro-apoptotic and antiapoptotic response of keratinocytes (KC) to UV light is of particular relevance to the development of skin cancer (Brash et al, 1996). Whereas both squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) arise from epidermal KC, it is becoming increasingly apparent that the natural history of their development, their underlying molecular pathogenesis, and potential involvement of antiapoptotic pathways are significantly different. Nonetheless, as pointed out later in the text, significant progress is being made in our understanding of the pathophysiology of these relatively common epithelial-cell-derived neoplasms. In this review we will explore four topics: first, a review of the life and death signaling pathways operative in normal human skin that prevents premature apoptosis of KC with an emphasis on nuclear factor kappaB (NFkappaB) survival signals; second, the molecular pathways that are engaged and regulate apoptosis after normal KC are exposed to ultraviolet (UV) light; third, the apoptotic resistant mechanisms that premalignant and malignant KC utilize to avoid cell death; fourth, therapeutic strategies that can render malignant cells more susceptible to apoptosis with an emphasis on a death pathway mediated by the death ligand TRAIL.

Cathepsin B is a lysosomal cysteine protease whose increased expression is believed to be linked to the malignant progression of tumors. Alternative splicing and the use of alternative transcription initiation sites in humans produce cathepsin B mRNAs that differ in their 5-and 3-untranslated ends. Some human tumors also contain cathepsin B-related transcripts that lack exon 3 which encodes the N-terminal signal peptide and 34 of the 62-amino acid inhibitory propeptide. In this study we show that one such transcript, CB(؊2,3), which is missing exons 2 and 3, is likely to be a functional message in tumors. Thus, CB(؊2,3) was found to be otherwise complete, containing the remainder of the cathepsin B coding sequence and the part of the 3-untranslated region that is common to all previously characterized cathepsin B mRNAs in humans. Its in vitro translation product can be folded to produce enzymatic activity against the cathepsin B-specific substrate, N ␣ -benzyloxycarbonyl-L-Arg-L-Arg-4-methylcoumaryl-7-amide. Endogenous CB(؊2,3) from the metastatic human melanoma cell line, A375M, co-sediments with polysomes, indicating that it engages the eukaryotic translation machinery in these cells. Epitope-tagged forms of the truncated cathepsin B from CB(؊2,3) are produced in amounts comparable to the normal protein after transient transfection into COS cells. Immunofluorescence microscopy and subcellular fractionation show this novel tumor form of cathepsin B to be associated with nuclei and other membranous organelles, where it is likely to be bound to the cytoplasmic face of the membranes. This subcellular distribution was different from the lysosomal pattern shown by the epitope-tagged, full-length cathepsin B in COS cells. These results indicate that the message missing exons 2 and 3 is likely to be translated into a catalytically active enzyme, and that alternative splicing (exon skipping) could contribute to the aberrant intracellular trafficking of cathepsin B that is observed in some human cancers.Cathepsin B is a lysosomal cysteine protease whose expression and trafficking are frequently altered in transformed and malignant cells (1). Both its association with the plasma membrane and its secretion have been linked to the increased capacity of human and rodent tumors to invade and metastasize (2-5). The association of cathepsin B with cell fractions containing plasma membrane-derived vesicles and endosomes was found to increase after transfection of a human breast epithelial cell line with the c-Ha-ras oncogene (6). Cathepsin B has been detected in nuclei from human ectocervical tumors and in basal and columnar cells of the normal and hyperplastic prostate (7,8). In human lung tumor cell lines cathepsin B activity was also localized to the endoplasmic reticulum, nuclear membrane, and plasma membrane (9). Forms of cathepsin B greater in size and stability than the mature, lysosomal form have been observed in tumors from humans and animals or in media conditioned by them (10 -13). At present, many of these findings ...

The impact of low-dose ultraviolet light (UV-light) on apoptotic susceptibility of keratinocytes (KCs) induced by TRAIL is unclear. Skin expresses a functional form of TRAIL, and while sun exposure influences TRAIL death receptors, a role for decoy receptors has not been evaluated. Unraveling mechanisms involving apoptotic sensitivity of KCs is important because skin is the first target of UV-light, and a site for commonly occurring cancers. Since apoptosis is a homeostatic process eliminating UV-light induced DNA damaged cells, elucidating molecular events regulating apoptosis enhances understanding of cutaneous photocarcinogenesis. Here we demonstrate low-dose UV-light enhances susceptibility of KCs to TRAIL-induced apoptosis. Low-dose UV-light selectively reduces decoy receptors, without influencing death receptor levels. UV-induced enhanced apoptotic susceptibility was reduced by over-expression of decoy receptor TRAIL-R4, but not TRAIL-R3; or treatment with thiol compound pyrrolidine dithiocarbamate (PDTC), which also enhanced TRAIL-R4 levels. Besides influencing decoy receptors, low-dose UV-light plus TRAIL also synergistically promoted cytochrome c and Smac release from mitochondria. Inhibitors directed against caspases 2, 3, 8, and 9 reduced the synergistic apoptotic response following low-dose UV-light plus TRAIL exposure; as did forced over-expression of Bcl-x and dominant negative (DN) constructs of FADD and caspase 9. Thus, relative levels of decoy receptors significantly influence susceptibility of KCs to TRAIL-induced apoptosis with concomitant low-dose UV-light exposure; in addition to the apoptotic pathway mediated by mitochondrial permeabilization.

A new member of the gamma-herpesvirus family, HHV-8 (also known as Kaposi's sarcoma (KS)-associated herpesvirus), has been linked to KS and body cavity-based lymphoma. Other members of this family, eg, Epstein-Barr virus, were originally thought to have only one strain, but subsequent analysis revealed different strains correlating to cellular patterns of infectivity and geographical location. To determine whether multiple strains of HHV-8 exist, we compared DNA sequences among KS and body cavity-based lymphoma-derived HHV-8 and examined differences in HHV-8 subgroups between American and Saudi Arabian iatrogenic KS patients. Samples were analyzed by polymerase chain reaction using multiple primer sets to five different open reading frames from HHV-8, and DNA sequencing was performed. HHV-8 DNA was present in all of our KS and body cavity-based lymphoma samples by polymerase chain reaction. HHV-8 DNA was detected in each body cavity-based lymphoma sample using a majority of the primers, whereas only two primer sets consistently amplified HHV-8 DNA derived from KS lesions. DNA sequencing within open reading frames 26 and 27 indicate the existence of at least three variants of HHV-8, with the majority of iatrogenic KS patients in Saudi Arabia containing unique nucleotide changes that may define a distinct, previously unidentified subgroup we term SA, whereas those from America were of Group A or B. Thus, although the sequencing data within open reading frames 26 and 27 did not permit discrimination between patients with lymphoma versus KS disease processes, HHV-8 derived from Saudi Arabian KS lesions were shown to have a distinct nucleotide sequence not seen in any of the other clinical samples examined.