Growing evidence indicates a central role for p53 in mediating cell cycle arrest in response to mitotic spindle defects so as to prevent rereplication in cells in which the mitotic division has failed. Here we report that a transient inhibition of spindle assembly induced by nocodazole, a tubulin-depolymerizing drug, triggers a stable activation of p53, which can transduce a cell cycle inhibitory signal even when the spindle-damaging agent is removed and the spindle is allowed to reassemble. Cells transiently exposed to nocodazole continue to express high levels of p53 and p21 in the cell cycle that follows the transient exposure to nocodazole and become arrested in G 1 , regardless of whether they carry a diploid or polyploid genome after mitotic exit. We also show that p53 normally associates with centrosomes in mitotic cells, whereas nocodazole disrupts this association. Together these results suggest that the induction of spindle damage, albeit transient, interferes with the subcellular localization of p53 at specific mitotic locations, which in turn dictates cell cycle arrest in the offspring of such defective mitoses.

Peptide trimming in the endoplasmic reticulum (ER), the final step required for the generation of most HLA class I-binding peptides, implicates the concerted action of two aminopeptidases, ERAP1 and ERAP2. Because defects in the expression of these peptidases could lead to aberrant surface HLA class I expression in tumor cells, we quantitatively assayed 14 EBV-B cell lines and 35 human tumor cell lines of various lineages for: 1) expression and enzymatic activities of ERAP1 and ERAP2; 2) ER peptide-trimming activity in microsomes; 3) expression of HLA class I H chains and TAP1; and 4) surface HLA class I expression. ERAP1 and ERAP2 expression was detectable in all of the EBV-B and tumor cell lines, but in the latter it was extremely variable, sometimes barely detectable, and not coordinated. The expression of the two aminopeptidases corresponded well to the respective enzymatic activities in most cell lines. A peptide-trimming assay in microsomes revealed additional enzymatic activities, presumably contributed by other unidentified aminopeptidases sharing substrate specificity with ERAP2. Interestingly, surface HLA class I expression showed significant correlation with ERAP1 activity, but not with the activity of either ERAP2 or other unidentified aminopeptidases. Transfection with ERAP1 or ERAP2 of two tumor cell lines selected for simultaneous low expression of the two aminopeptidases resulted in the expected, moderate increases of class I surface expression. Thus, low and/or imbalanced expression of ERAP1 and probably ERAP2 may cause improper Ag processing and favor tumor escape from the immune surveillance.

Neuroblastoma (NB) is the most common solid extracranial cancer of childhood. Amplification and overexpression of the MYCN oncogene characterize the most aggressive forms and are believed to severely downregulate MHC class I molecules by transcriptional inhibition of the p50 NF-κB subunit. In this study, we found that in human NB cell lines, high MYCN expression is not responsible for low MHC class I expression because neither transfection-mediated overexpression nor small interfering RNA suppression of MYCN affects MHC class I and p50 levels. Furthermore, we identified NF-κB as the immediate upstream regulator of MHC class I because the p65 NF-κB subunit binds MHC class I promoter in chromatin immunoprecipitation experiments, and MHC class I expression is enhanced by p65 transfection and reduced by (a) the chemical NF-κB inhibitor sulfasalazine, (b) a dominant-negative IKBα gene, and (c) p65 silencing. Moreover, we showed that the endoplasmic reticulum aminopeptidases ERAP1 and ERAP2, which generate MHC class I binding peptides, are regulated by NF-κB, contain functional NF-κB-binding elements in their promoters, and mimic MHC class I molecules in the expression pattern. Consistent with these findings, nuclear p65 was detected in NB cells that express MHC class I molecules in human NB specimens. Thus, the coordinated downregulation of MHC class I, ERAP1, and ERAP2 in aggressive NB cells is attributable to a low transcriptional availability of NF-κB, possibly due to an unknown suppressor other than MYCN. Cancer Res; 70(3); 916-24. ©2010 AACR.

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