Retinoblastoma protein regulates the crosstalk between autophagy and apoptosis, and favors glioblastoma resistance to etoposide.

Glioblastomas (GBMs) are devastating tumors of the central nervous system, with a poor prognosis of 1-year survival. This results from a high resistance of GBM tumor cells to current therapeutic options, including etoposide (VP-16). Understanding resistance mechanisms may thus open new therapeutic avenues. VP-16 is a topoisomerase inhibitor that causes replication fork stalling and, ultimately, the formation of DNA double-strand breaks and apoptotic cell death. Autophagy has been identified as a VP-16 treatment resistance mechanism in tumor cells. Retinoblastoma protein (RB) is a classical tumor suppressor owing to its role in G1/S cell cycle checkpoint, but recent data have shown RB participation in many other cellular functions, including, counterintuitively, negative regulation of apoptosis. As GBMs usually display an amplification of the EGFR signaling involving the RB protein pathway, we questioned whether RB might be involved in mechanisms of resistance of GBM cells to VP-16. We observed that RB silencing increased VP-16-induced DNA double-strand breaks and p53 activation. Moreover, RB knockdown increased VP-16-induced apoptosis in GBM cell lines and cancer stem cells, the latter being now recognized essential to resistance to treatments and recurrence. We also showed that VP-16 treatment induced autophagy, and that RB silencing impaired this process by inhibiting the fusion of autophagosomes with lysosomes. Taken together, our data suggest that RB silencing causes a blockage on the VP-16-induced autophagic flux, which is followed by apoptosis in GBM cell lines and in cancer stem cells. Therefore, we show here, for the first time, that RB represents a molecular link between autophagy and apoptosis, and a resistance marker in GBM, a discovery with potential importance for anticancer treatment.

Electron and video-light microscopy analysis of the in vitro effects of pyrantel pamoate on Giardia lamblia.

Electron and video-light microscopy analysis of the in vitro effects of pyrantel pamoate on Giardia lamblia. Experimental Parasitology 97, 9-14. Giardia infection is predominant in the small intestine of vertebrates, where the trophozoites attach to epithelial cells and adversely affect the microvilli and other epithelial cell structures. Giardiasis, the disease caused by this protozoan, is very common in developing countries and mainly affects children. Drugs currently used to treat Giardia infection, such as some benzimidazole derivatives, were originally designed to treat helminthic infections. Many of the drugs are known to cause severe side effects and disturbances to the patient. Using transmission electron microscopy and video-light microscopy, we studied the effects of pyrantel pamoate, a drug commonly used in the treatment of helminthic infections in horses and ruminants, on Giardia lamblia trophozoites. Pyrantel pamoate was administered to Giardia cells in four different concentrations. Using video-light microscopy, we observed the decrease in flagella beating frequency and severe changes in the lateral flange and in the general aspect of the cell. Using transmission electron microscopy, we observed changes in the cytoplasm and peripheral vesicles. The flagella and adhesive disk structure were not affected. Apparently, the effects of pyrantel pamoate are irreversible.

Expression of tubulin polyglycylation in Giardia lamblia.

By means of immunofluorescence, immunoelectron microscopy and immunoblotting, we show that polyglycylation, a posttranslational modification of tubulin widely spread among eukaryotes, is present in the diplomonad, Giardia lamblia, a putative ancestral cell possessing a highly developed microtubular cytoskeleton. This modification was recently discovered in the ciliated protist, Paramecium, and was not found in the Euglenozoa, a lineage considered as ancient. We used two monoclonal antibodies (mAbs), TAP 952 and AXO 49, specifically recognizing mono- and polyglycylated tubulin isoforms, to detect this modification in Giardia extracts and to localize it in the different classes of microtubules within the cell. The alpha- and beta-tubulin subunits were recognized by the two mAbs, indicating that both tubulin subunits are glycylated, in agreement with lately reported mass spectrometry results. Noticeably, Giardia tubulin was much more reactive with AXO 49 than with TAP 952. In situ, AXO 49 intensely labeled the microtubules present in the four pairs of flagella and the median body, and lightly decorated the microtubules from the adhesive disc. In contrast, TAP 952 intensely labeled only the microtubules of the median body. The results indicate a differential expression of glycylated isoforms within various microtubular structures of Giardia lamblia. They also suggest that the complete set of enzymes required for polyglycylation is expressed in very divergent eukaryotes.

Synaptophysin and synapsin I as tools for the study of the exo-endocytotic cycle.

Synaptophysin, an integral protein of the synaptic vesicle membrane, and synapsin I, a phosphoprotein associated with the cytoplasmic side of synaptic vesicles, represent useful markers that allow to follow the movements of the vesicle membrane during recycling. The use of antibodies against these proteins to label nerve terminals during experimental treatments which stimulate secretion has provided evidence that during the exo-endocytotic cycle synaptic vesicles transiently fuse with the axolemma, from which they are specifically recovered. When recycling is blocked, exocytosis leads to the permanent incorporation of the synaptic vesicle membrane into the axolemma and to diffusion of the vesicle components in the plane of the membrane.