Dendritic cells (DCs) that acquired antigen from apoptotic tumor cells are able to induce major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocytes and antitumor immunity. In the present study, we investigated the efficiency of antitumor immunity derived from DCs that had phagocytosed apoptotic/necrotic BL6-10 melanoma cells compared with that of DCs pulsed with the tumor mTRP2 peptide. Our data showed that phagocytosis of apoptotic/ necrotic tumor cells resulted in maturation of DCs with up-regulated expression of proinflammatory cytokines [interleukin (IL)-1␤, IL-6, tumor necrosis factor-␣, interferon-␥ and granulocyte-macrophage colony-stimulating factor], chemokines (MIP-1␣, MIP-1␤ and MIP-2), the CC chemokine receptor CCR7 and the cell surface molecules (MHC class II, CD11b, CD40 and CD86), and down-regulated expression of the CC chemokine receptors CCR2 and CCR5. These mature DCs displayed enhanced migration toward the CC chemokine MIP-3␤ in a chemotaxis assay in vitro and to the regional lymph nodes in an animal model in vivo. Our data also showed that vaccination with DCs that had phagocytosed apoptotic/necrotic BL6-10 cells was able to (i) more strongly stimulate allogeneic T-cell proliferation in vitro, (ii) induce an in vivo Th1-type immune response leading to more efficient tumor-specific cytotoxic CD8 ؉ T-cell-mediated immunity and (iii) eradicate lung metastases in all 6 vaccinated mice compared with mice vaccinated with DCs pulsed with the tumor mTRP2 peptide, in which lung metastases were reduced (mean number of 16 per mouse) but not completely eradicated. Therefore, DCs that had phagocytosed apoptotic/necrotic tumor cells appear to offer new strategies in DC cancer vaccines. © 2001 Wiley-Liss, Inc. Key words: cancer vaccine; dendritic cell maturation; apoptotic/ necrotic tumor cellCytotoxic T lymphocytes (CTLs) play a major role in the rejection of immunogenic tumors. 1 Classically, CTLs target tumors through recognition of a ligand consisting of the self major histocompatibility complex (MHC) class I molecule and peptides that are generally derived from tumor antigens synthesized within the tumor cells. 2,3 However, there is evidence for an exogenous pathway whereby antigens that are not expected to gain access to the cytoplasm are presented on MHC class I molecules. 4 -6 A most striking example of this is the in vivo phenomenon of crosspriming: antigens from donor cells are acquired by host antigenpresenting cells (APCs) and presented on MHC class I molecules in the appropriate context of co-stimulation. Delivery of exogenous antigen to the endogenous MHC class I-restricted processing pathway of APCs is a critical challenge in cancer vaccine design.Dendritic cells (DCs) are one of the most potent APCs. They capture antigens in situ, and migrate to lymphoid organs to interact/activate naive T cells. 7 DCs pulsed with synthetic tumorderived MHC class I-restricted peptides or tumor lysates and tumor cell-derived RNA induced significant CTL-dependent antitumor immune respon...

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These in vitro tests may prove valuable to screen potential tumor growth promoters.

R-Deprenyl and R-2-heptyl-N-methylpropargylamine (R-2-HMP) are compounds that have been shown to reduce neuronal death in various in vitro and in vivo models involving apoptosis but do not always prevent apoptosis. In the present study we have examined the effects of these compounds and their S enantiomers on cytosine arabinoside (ara C)-induced apoptosis and low K~-inducedapoptosis in cerebellar granule cells in primary culture. It was found that R-deprenyl and R-2-HMP could prevent ara C-induced apoptosis with an EC 50 around iO-~M but could not prevent low K~-induced apoptosis. S-Deprenyl and S-2-HMP did not prevent apoptosis under any conditions but were found to antagonize the antiapoptotic actions of R-deprenyl and R-2-HMP. Using the fluorescent mitochondrial dye chioromethyltetramethylrhodamine methyl ester it was found that there was a loss of mitochondrial function in cerebelar granule cells exposed to ara C but not low Kmedium. R-Deprenyl and R-2-HMP prevented the ara C-induced loss of mitochondrial function. It is concluded that Rdeprenyl and R-2-HMP prevent apoptosis of cerebetlar granule cells by a mechanism that is independent of monoamine oxidase inhibition and that they act on the same site to prevent specifically apoptosis involving a loss of mitochondrial membrane potential, possibly p53-dependent apoptosis.

We have previously shown that UEV1 is up-regulated in all tumor cell lines examined and when SV40-transformed human embryonic kidney cells undergo immortalization; however, it is unclear whether and how UEV1 plays a critical role in this process. UEV1A encodes a ubiquitin conjugating enzyme variant, which is required for Ubc13 (ubiquitin conjugating enzyme) catalyzed poly-ubiquitination of target proteins through Lys63-linked chains. One of the target proteins is NEMO/IKKgamma (nuclear factor-kappaB essential modulator/inhibitor of kappaB protein kinase), a regulatory subunit of IkappaB kinase in the NF-kappaB signaling pathway. In this report, we show that constitutive high-level expression of UEV1A alone in cultured human cells was sufficient to cause a significant increase in NF-kappaB activity as well as the expression of its target anti-apoptotic protein, Bcl-2 (B-cell leukemia/lymphoma 2). Overexpression of UEV1A also conferred prolonged cell survival under serum-deprived conditions, and protected cells against apoptosis induced by diverse stressing agents. All of the effects of Uev1A were reversible upon suppression of UEV1 expression by RNA interference. Our observations presented in this report provide evidence that Uev1A is a critical regulatory component in the NF-kappaB signaling pathway in response to environmental stresses and identify UEV1A as a potential proto-oncogene.