Erzsébet Fellinger

RELATED ORGANELLES OF THE ENDOSOME-LYSOSOME SYSTEM CONTAIN A DIFFERENT REPERTOIRE OF UBIQUITINATED PROTEINS IN SF9 INSECT CELLS

Two components of the endosomal/lysosomal compartment of Sf9 cells, multivesicular bodies (MVB) and light vacuoles with membrane complexes (LVMC) have been isolated and probed for ubiquitin protein conjugates with a specific antibody. Immunogold electron microscopy indicates that whereas ubiquitin‐protein conjugates are localised to electron dense areas of MVB they are associated with the membranes of LVMC. Five ubiquitinated polypeptides are revealed in MVB by immunoblotting while numerous ubiquitinated species forming a smear following electrophoresis are present in LVMC. We suggest two possible routes for entry of ubiquitin‐protein conjugates into these organelles, via the cell surface and via primary lysosomes.

Dynamics of vinblastine-induced autophagocytosis in murine pancreatic acinar cells: Influence of cycloheximide post-treatments

Accumulation of autophagic vacuoles (AVs) was monitored by electron microscopic morphometry in murine pancreatic acinar cells during the 5-hr period after a single injection of vinblastine (VBL). The expansion of the autophagic compartment (AC) occurred in two waves. AVs accumulated in the first 90 min and regressed in the next hour, but thereafter AC expanded again, and 5 hr following the VBL injection, as much as 5.3% of the cytoplasmic volume was found sequestered into the AC. The high rates of accumulation of AVs indicated that VBL stimulated AV formation (segregation) during both expansion phases. To have a deeper insight into the dynamics of the process segregational inhibitor cycloheximide (CHI) was given 1 and 3 hr after VBL and the subsequent regression of the AC and its subcompartments (i.e., early, advanced, and late AVs) were measured during the next 90 min. We found that regression of AVs was fast in the first expansion and slowed down in the second expansion phase during which only early AVs regressed. CHI proved to be a fast and effective inhibitor of autophagic segregation, whether it was given before, simultaneously, or after the VBL injection. The aforementioned results argue for a dual mode of action of VBL (i.e., a prompt stimulation of segregation and a delayed retardation of AV maturation). The two effects of the alkaloid prevail differently along the time course. A further analysis of the behavior of the AC subcompartments showed that CHI perhaps inhibits segregational step(s) occurring prior to the actual formation of the autolysosomes.

A New Daunomycin–Peptide Conjugate: Synthesis, Characterization and the Effect on the Protein Expression Profile of HL-60 Cells in Vitro

Daunomycin (Dau) is a DNA-binding antineoplastic agent in the treatment of various types of cancer, such as osteosarcomas and acute myeloid leukemia. One approach to improve its selectivity and to decrease the side effects is the conjugation of Dau with oligopeptide carriers, which might alter the drug uptake and intracellular fate. Here, we report on the synthesis, characterization, and in vitro biological properties of a novel conjugate in which Dau is attached, via an oxime bond, to one of the cancer specific small peptides (LTVSPWY) selected from a random phage peptide library. The in vitro cytostatic effect and cellular uptake of Dau═Aoa-LTVSPWY-NH(2) conjugate were studied on various human cancer cell lines expressing different levels of ErbB2 receptor which could be targeted by the peptide. We found that the new daunomycin-peptide conjugate is highly cytostatic and could be taken up efficiently by the human cancer cells studied. However, the conjugate was less effective than the free drug itself. RP-HPLC data indicate that the conjugate is stable at least for 24 h in the pH 2.5-7.0 range of buffers, as well as in cell culture medium. The conjugate in the presence of rat liver lysosomal homogenate, as indicated by LC-MS analysis, could be degraded. The smallest, Dau-containing metabolite (Dau═Aoa-Leu-OH) identified and prepared expresses DNA-binding ability. In order to get insight on the potential mechanism of action, we compared the protein expression profile of HL-60 human leukemia cells after treatment with the free and peptide conjugated daunomycin. Proteomic analysis suggests that the expression of several proteins has been altered. This includes three proteins, whose expression was lower (tubulin β chain) or markedly higher (proliferating cell nuclear antigen and protein kinase C inhibitor protein 1) after administration of cells with Dau-conjugate vs free drug.

Time course of the quantitative changes in the autophagic-lysosomal and secretory granule compartments of murine liver cells under the influence of vinblastine

SummaryThe dynamics of the transient expansion of the autophagic-lysosomal (ALC) and secretory granule (SGC) compartments in mouse liver cells were monitored by electron microscopic morphometry after a single injection of 10 mg/kg b.w. vinblastine sulfate (VBL). Initially (first phase) the cytoplasmic volume fractions of the total ALC and its subcompartments, as well as of the SGC increased by an order of magnitude and peaked at the second h. In thesecond phase, all the aformentioned compartments regressed gradually, approaching their normal size between 12 and 36 h after VBL injection. Analysis of the dynamic changes in fractional volumes of subcompartments of the ALC showed that early autophagic vacuoles (AV1) were the first to enlarge. Advanced AVs (AV2) reacted 30 min later and a further 30 min time lag was required before late autolysosomes, appearing as dense bodies (DB), started to expand. We regard these data as kinetic proof that the bodies of the later reacting subcompartments developed from the earlier reacting ones. The time lag between the expansion of AV1 and AV2 subcompartments may be explained by a period of retardation of conversion of nascent autophagosomes (AV1) to autolysosomes (AV2) which is known to occur normally by fusion of AV1 with enzymecarrying lysosomes. However, transformation of AV1 to AV2 and later to DB resumed after the respective time lags. Moreover, our quantitative data lend support to the view that segregation of cytoplasmic portions into newly-formed autophagosomes was stimulated by VBL, at least in the first 2 h of treatment. The expansion of ALC accelerated during this period and led to an obvious overload of the lysosomal apparatus. DNA-based specific activity of the lysosomal marker enzyme acid phosphatase did not alter significantly during the 36 h period. The volume fraction of the SGC reacted to VBL in essentially the same way as the ALC. The possible mechanisms underlying the time course of the volume changes of ALC and SGC are briefly discussed.

Combined effects of fasting and vinblastine treatment on serum insulin level, the size of autophagic-lysosomal compartment, protein content and lysosomal enzyme activities of liver and exocrine pancreatic cells of the mouse

1. The volume fraction of autophagic vacuoles in liver parenchymal and exocrine pancreatic cells was smallest and the serum insulin level highest in the 24 hr prestarved mouse immediately after 3 hr feeding period. 2. The size of the autophagic vacuole and lysosome (dense body) compartments increased in both types of cells during 2-72 hr fasting parallel with decreasing serum insulin levels. 3. The protein content of the cells decreased and the DNA-based activity of acid phosphatase showed little change throughout fasting. The activity of cathepsin D increased during days 2 and 3 of food deprivation. 4. Vinblastine (50 mg/kg body wt) applied for the last 2 hr of different periods (2, 12, 24, 48 and 72 hr) of fasting decreased serum insulin level and increased the fractional cytoplasmic volume of autophagic vacuoles and dense bodies. This increase was smaller when the drug was applied shortly after feeding and much larger after prolonged fasting. The increase was more pronounced in the pancreatic than in the liver cells. 5. Our data show that the effect of vinblastine on the size of the autophagic-lysosomal compartment depends on the feeding status of the animals.