Dellis (Orsay, France)
Dellis (Orsay, France). cells. A treatment with chloroquine, an inhibitor of autophagy, potentiated the apoptotic effect of nutlin-3, particularly in those EBV-positive cells which were resistant to apoptosis induced by nutlin-3 alone, thereby showing that autophagy participates in this resistant phenotype. Finally, using immunohistochemical staining, clinical samples from various B cell lymphoproliferations with the EBV-positive latency II or III phenotype were found to harbor a constitutively active autophagy. gene promoter,24 we then tested the same cell lines for expression of the BECN1 protein which was found to follow that of RELA (Fig. 2A and Fig. S1). To examine RELA expression levels more precisely, cytosolic and nuclear extracts were prepared from both EBV-positive latency III and EBV-negative cell lines. Levels of RELA were found to be higher in the nuclear fraction of EBV-positive cell lines than in their EBV-negative counterparts, contrasting with the cytosolic GGTI298 Trifluoroacetate fractions where no such relation was observed (Fig. S2). This is consistent with RELA playing a role in the process leading to BECN1 expression based on its transcriptional regulatory function. GGTI298 Trifluoroacetate To confirm that LMP1 regulates BECN1 expression through the NFKB pathway we used stable transfectants of DG75 cells, which express LMP1 only in the absence of tetracycline. In these conditions of LMP1 expression, levels of both RELA and BECN1 increased as compared to control cells cultivated in the presence of tetracycline (Fig. 2B). We also used an shRNA approach to test for a direct correlation between the status of the NFKB-BECN1 pathway and the level of autophagy in EBV-positive latency III cells. To this end, RPMI8866 cells Mouse monoclonal to CD45RO.TB100 reacts with the 220 kDa isoform A of CD45. This is clustered as CD45RA, and is expressed on naive/resting T cells and on medullart thymocytes. In comparison, CD45RO is expressed on memory/activated T cells and cortical thymocytes. CD45RA and CD45RO are useful for discriminating between naive and memory T cells in the study of the immune system were transduced with an shRNA directed against and the levels of expression of RELA, BECN1, LC3-I and LC3-II were tested. As seen in Figure 2C, levels of BECN1 and LC3-II were found strongly decreased in transduced cells where RELA expression was virtually GGTI298 Trifluoroacetate abolished as compared to control cells transduced with an shRNA that does not target any known human gene. LC3-I expression was not affected by inhibition of RELA. Altogether, these data indicate that an LMP1-dependent activation of the NFKB signaling pathway upregulates the expression of BECN1 and the level of autophagy in EBV-positive latency III cells. Open in a separate window Figure 2. RELA activation and BECN1 expression in EBV-negative and EBV-positive latency III lymphoid cell lines. (A) Whole cell lysates were analyzed by western blotting for RELA and BECN1 expression. (B) Whole cell lysates prepared from DG75 cells, expressing LMP1 in a tetracycline-regulated system, were tested for expression of LMP1, RELA and BECN1. (C) Whole cell lysates prepared from RPMI8866 stably transduced with a (fold change 6.9 and 13.3), (Sestrin 2) (fold change 5.2 and 9.2), (tuberous sclerosis 2) (fold change 2, only in BL2/B95). The proteins encoded by these genes are involved in a cascade of events: AMPK is activated by direct interactions with SESN1 and SESN2 and phosphorylates TSC2 which, in turn, inhibits MTOR thus leading to autophagy activation.5 Treatment of BL2 and BL2/B95 with nutlin-3 did not modify the mRNA level of (data not shown). Open in a separate window Figure 3. Changes in global gene expression analysis of EBV-negative BL2 and EBV-positive latency III BL2/B95 cells according to nutlin-3 treatments. (A) Probes corresponding to genes relative to autophagy are represented using heatmaps. (B) Fold change in mRNA levels for autophagy-related genes in both cell lines treated for 16?h as compared to untreated cells. Treatment with nutlin-3 promotes autophagy in EBV-positive latency III cells but not in EBV-negative cells Since nutlin-3 treatment increases the level of expression of several genes involved in autophagy, we decided to assess if this compound is GGTI298 Trifluoroacetate able to increase the autophagic flux in EBV-negative.