Alzheimer's disease (AD) is caused by the cerebral deposition of ␤-amyloid (A␤), a 38 -43-amino acid peptide derived by proteolytic cleavage of the amyloid precursor protein (APP). Initial studies indicated that final cleavage of APP by the ␥-secretase (a complex containing presenilin and nicastrin) to produce A␤ occurred in the endosomal/lysosomal system. However, other studies showing a predominant endoplasmic reticulum localization of the ␥-secretase proteins and a neutral pH optimum of in vitro ␥-secretase assays have challenged this conclusion. We have recently identified nicastrin as a major lysosomal membrane protein. In the present work, we use Western blotting and immunogold electron microscopy to demonstrate that significant amounts of mature nicastrin, presenilin-1, and APP are co-localized with lysosomal associated membrane protein-1 (cAMP-1) in the outer membranes of lysosomes. Furthermore, we demonstrate that these membranes contain an acidic ␥-secretase activity, which is immunoprecipitable with an antibody to nicastrin. These experiments establish APP, nicastrin, and presenilin-1 as resident lysosomal membrane proteins and indicate that ␥-secretase is a lysosomal protease. These data reassert the importance of the lysosomal/endosomal system in the generation of A␤ and suggest a role for lysosomes in the pathophysiology of AD.

Using a unique proteomics approach in a large scale, cross-sectional cohort study, we identified elafin/trappin-2 as a novel innate immune factor, which is highly associated with resistance. This association was confirmed within an independent, prospective cohort study. Genital tract elafin/trappin-2 levels constitute a natural correlate of HIV protection in humans.

While melanomas are resistant to the cytotoxic effects of radiotherapy, little is known about the molecular mechanisms underlying this intrinsic resistance. Here, we describe the utilization of retroviral insertional mutagenesis to facilitate the analysis of genetic changes that are associated with radioresistance in human melanoma. A radial growth phase human melanoma cell line, WM35, was infected with a replication-defective amphotropic murine retrovirus and subsequently selected for X-ray radiation-resistant variants. Several radiation-resistant clones were independently isolated and characterized. Interestingly, these clones also displayed resistance to ultraviolet radiation and to the chemotherapeutic drug cisdiamminedichloroplatinum(II) (CDDP). By Northern and Western analyses, we showed that the expression of DOPAchrome tautomerase (DCT), also known as tyrosinase-related protein 2 (TYRP2), an enzyme that functions in eumelanin synthesis, was significantly elevated in the radiation-resistant clones relative to the parental WM35 cells. Moreover, the levels of DCT in a variety of human melanoma cell lines correlated with their relative levels of radioresistance and the enforced expression of DCT conferred increased resistance to UV (B) treatment. An analysis of stress signaling induced by radiation as well other cytotoxic stressors showed that resistance associated with DCT overexpression applied specifically to treatments that activate the ERK/MAPK pathway. Indeed, DCT overexpression in a melanoma cell line resulted in increased ERK activity. Moreover, ectopic expression of dominant-active MEK in this melanoma cell line conferred UV(B) resistance suggesting that the ERK/ MAPK pathway downstream of DCT may play a critical role in radiation and drug resistance. Overall, given that each gamma-and UV(B)-resistant cell line also exhibited resistance to CDDP and that CDDP-resistant clones showed increased resistance to UV(B) irradiation, these results suggest a common mechanism underlying radioand chemoresistance, which is mediated by DCT expression.

A major obstacle in the systemic treatment of advanced malignant melanoma is its intrinsic resistance to conventionally used chemotherapeutic agents. In order to investigate the mechanisms of this intrinsic resistance, we have previously utilized retroviral insertional mutagenesis on an early-stage, drug sensitive human melanoma cell line (WM35) to establish mutated cell lines that exhibited increased resistance to cis-diamminedichloroplatinum(II) (CDDP). Here, we demonstrate that this increased resistance to CDDP is mediated by the over-expression of tyrosinase-related protein-2 (TYRP2), an enzyme that normally functions in the biosynthesis of the pigment, melanin. Northern and Western blot analyses revealed that the expression of TYRP2 in the virally-derived cell lines as well as in a panel of human melanoma cell lines positively correlated with their levels of resistance to CDDP. Furthermore, enforced expression of TYRP2 in WM35 cells by transfection elevated their resistance to CDDP. The increased CDDP resistance in the virally-derived clones and TYRP2 transfectants was accompanied by a reduction in CDDP-induced apoptosis. Interestingly, the virally-derived CDDP-resistant clones also showed cross resistance to carboplatin and methotrexate, but not taxol, suggesting that TYRP2 over-expression may confer resistance speci®cally to DNA damaging agents.Overall, these results demonstrate a novel mechanism of drug resistance in human melanoma cells that is mediated by the over-expression of TYRP2. Since TYRP2 is expressed only in cells of melanocytic lineage, this may represent the ®rst report of a lineage-speci®c mechanism of drug resistance. In summary, these ®ndings suggest a signi®cant role for TYRP2 in the intrinsic drug resistance phenotype of human melanoma cells and may have important implications in the development of chemosensitization strategies for the clinical management of this disease. Oncogene (2000) 19, 395 ± 402.