Although overexpression of the low-affinity p75 neurotrophin receptor (p75NTh) is frequently associated with advanced stages of human melanoma progression, the functional significance of this finding is unknown. We examined whether the degree of cell surface expression of p75NTR in human melanoma cell variants determines their extent of invasion stimulated by nerve growth factor (NGF). Treatment of MeWo melanoma cells or a metastatic spontaneous wheat germ agglutinin-resistant variant subline (70W) of MeWo cells with 2.5S NGF resulted in a dose-dependent enhancement of invasion through a reconstituted basement membrane. This effect was most pronounced with the 70W subline that exhibits brain-metastasizing potential in nude mice but was not found with a poorly metastatic MeWo variant subline (3S5). The expression of p75NTR as determined by Northern blotting and immunoprecipitation analysis of 1251I-labeled cell surface proteins correlated with NGFstimulated invasion. The MeWo melanoma sublines used in this study did not express pl4OPrt°otr" mRNA or any p140Proto-trk variant transcripts including p70tr`as determined by Northern analysis and RT-PCR analysis. Thus, these melanoma cells would not be expected to form functional p75-p140 heterodimers or p140-p140 homodimers capable of transducing an NGF-generated signal to pl4OProto-tr cytoplasmic substrates. These cells did express authentic pl45trkc transcripts. However, NGF did not catalytically activate pl45t receptors via increased tyrosine phosphorylation as would be expected if pl45trkc participated in the signaling established by NGF. Furthermore, a NGF-stimulated wurineanalogue-sensitive kinase activity was found to coimmunoprecipitate with p75N . This p75 -associated kinase may coordinate initial signaling events evoked by p75NTR ligand interaction. Addition of 2.5S NGF, at concentrations that should saturate cell surface p75NTR, to matrix-adherent cultures of human MeWo and 70W but not 3S5 melanoma cells suppressed the expression of 92-kDa type IV collagenase and stimulated the production of 72-kDa type IV collagenase in its fully active 68-kDa form. In the absence of pl40Proto-trkA, the matrix-dependent effects of NGF on metalloproteinase expression of brain-metastatic 70W melanoma cells suggest a signaling role for the low-affinity melanoma p75NTR receptor and its associated purine-analogue-sensitive kinase in signaling enhanced matrix penetration of NGF-rich stromal microenvironments such as the brain.

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Bcl-2 is an integral membrane oncoprotein that localizes to membranes of the mitochondria, endoplasmic reticulum, and nuclear envelope. Bcl-2 is a member of a family of cell death regulators and functions to inhibit apoptosis. Using confocal microscopy and immunoblotting we show that the ability of bcl-2 to suppress cell death following genotoxic damage can be a consequence of inhibiting nuclear import of induced wild-type p53 protein. Our data suggests that the ability of bcl-2 to modulate tra cking events is not cell type speci®c. These data support a`gatekeeper' mechanism for cell death suppression by bcl-2.

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