Dynamics of microtubules, motor proteins and 20S proteasomes during bovine oocyte IVM.

The present study investigated the distribution of cytoplasmic dynein, dynactin and 20S proteasomes in oocytes isolated from small (<2 mm) and large (2-8 mm) follicles during IVM. Immediately after chromatin condensation (germinal vesicle (GV) breakdown), dynactin was closely associated with the chromatin and interacted with tubulin at the MI and MII spindles in oocytes recovered from large follicles. Dynactin showed perinuclear concentration. Dynein was homogeneously distributed in the cytoplasm of GV oocytes in both groups and was associated with the chromatin at the MI and MII spindle. The 20S proteasomes were found predominantly in the nucleus at the GV stage and were associated with the chromatin up to the MII stage in both groups of oocytes. The use of sodium orthovanadate, an inhibitor or phosphatase and ATPase activity, and nocodazole, a known disruptor of microtubules, affected the localisation of proteasomes in the meiotic stages. The results demonstrate the distinct dynamics of molecular motors and proteasomes during bovine oocyte IVM, their possible relationship with the developmental competence of the oocyte and the link between microtubules, their associated molecular motors and the transport of proteasomes during bovine female meiosis.

Increased local vascular endothelial growth factor expression associated with antitumor activity of proteasome inhibitor.

Inhibition of the proteasome, a multicatalytic proteinase complex, is an attractive approach to cancer therapy. Here we report that a selective inhibitor of the chymotrypsin-like activity of the proteasome, PSI (N-benzyloxycarbonyl-Ile-Glu(O-t-butyl)-Ala-leucinal) may inhibit growth of solid tumors not only through apoptosis induction, but also indirectly--through inhibition of angiogenesis. Two murine tumors: colon adenocarcinoma (C-26) and Lewis lung carcinoma (3LL) were chosen to study the antitumor effect of PSI. In an in vivo model of local tumor growth, PSI exerted significant antitumor effects against C-26 colon carcinoma, but not against 3LL lung carcinoma. Retardation of tumor growth was observed in mice treated with both 10 nmoles and 100 nmoles doses of PSI and in the latter group prolongation of the survival time of tumor-bearing mice was observed. PSI inhibited angiogenesis in the C-26 growing tumors with no such effect in 3LL tumors. Unexpectedly, that activity was associated with upregulation of vascular endothelial growth factor (VEGF) at the level of mRNA expression and protein production in C-26 tumors treated with PSI. C-26 cells treated with PSI produced increased amounts of VEGF in vitro in a dose- and time-dependent manner. We demonstrated that in C-26 colon adenocarcionoma higher VEGF production may render endothelial cells susceptible to the proapoptotic activity of PSI and is associated with inhibition of tumor growth.

Effects of the combination of a proteasome inhibitor (PSI) and an inhibitor of ubiquitin-ligases (Leu-Ala) on the ultrastructure of human leukemic U937 cells.

We have used the dipeptide Leu-Ala in an attempt to prevent the formation of ubiquitin-protein conjugates in U937 cells by inhibition of cellular E3 enzymes (ubiquitin ligases). Proteasome inhibitors induce the formation of perinuclear aggregates of ubiquitinated proteins and proteasomes (aggresomes) in the area of the proteolytic center of the cell. Leu-Ala did not prevent the forrmation of those aggregates under the action of PSI (peptidyl aldehyde, selective inhibitor of the chymotrypsin-like activity of the proteasome), however it induced an accumulation of lipid droplets in treated cells, suggesting a previously unknown involvement of Leu-Ala in lipid metabolism. We conclude, that either Leu-Ala is not able to completely inhibit the cellular E3 enzymes or some of those enzymes are insensitive to this dipeptide, allowing therefore the build-up of ubiquitin-conjugates in the proteolytic centre of the cell.

Proteasome localization and ultrastructure of spermatozoa from patients with varicocele--immunoelectron microscopic study.

Localization of proteasomes in spermatozoa from patients with varicocele-associated sterility was studied by means of immunolabeling with the MPC21 monoclonal antibody detecting the C3 subunit of the 20S proteasome. The reaction was visualized for electron microscopy using the secondary Nano-Gold-coupled antibody with Gold-Enhancement in pre-embedding technique. We found that semen samples from varicocele patients contained a large amount of abnormal spermatozoa characterized by the presence of dispersed chromatin and large residual bodies (cytoplasmic droplets) as well as spermatids at various stages of spermiogenesis. In normal spermatozoa, the immunolabeling was found in the acrosome, postacrosomal regions, nuclear vacuoles, in the neck and in the middle-piece as well as in the residual bodies, while chromatin remained unlabeled. In varicocele spermatozoa, the immunolabeling was also associated with chromatin and large residual bodies (cytoplasmic droplets). In contrast to normal, mature spermatozoa, the chromatin of the cells at earlier stages of spermiogenesis was strongly immunolabeled. The association of proteasomes with sperm chromatin and large residual bodies can be the sign of abnormality and disturbances in spermatogenesis associated with varicocele.

Augmented pro-apoptotic effects of TRAIL and proteasome inhibitor in human promonocytic leukemic U937 cells.

TRAIL, Tumor necrosis factor-related apoptosis-inducing ligand), a member of the TNF family, is known to be cytotoxic for a high proportion of tumor cell lines. However, successful application of TRAIL in tumor therapy may depend on finding other agents that can potentiate its antitumor effects. The present study showed that the cytostatic/cytotoxic TRAIL activity against U937 cells could be significantly augmented by proteasome inhibitor PSI, as revealed by MTT assay. Increased cytostatic/cytotoxic effect on U937 cells by TRAIL/PSI combined treatment was caused by apoptosis, as shown by an increased PARP cleavage rate. TRAIL/PSI did not affect the level of mRNA expression for TRAIL receptors (DR4, DR5, DcR1) and other apoptosis signal transduction molecules (TRADD, caspase-8).

Lactacystin inhibits cathepsin A activity in melanoma cell lines.

We describe the inhibitory effect of the proteasome inhibitor, lactacystin, on cathepsin A activity in murine melanoma cell lines. In vitro lactacystin metabolite, beta-lactone, at a concentration of 1 microM, significantly suppressed cathepsin A activity in B78 melanoma cell lysates by about 50%. Exposure of three murine melanoma cell lines with different metastatic potential to lactacystin at a concentration of 5 microM for 6 h caused a significant reduction in the carboxypeptidase activity of this enzyme, while the inhibitory activity remained unchanged for at least 12 h. Other proteasome-specific inhibitors, e.g. epoxomicin and N-benzyloxycarbonyl-Ile-Glu(O-tert-Bu)-Ala-leucinal (PSI) at a concentration of 1 microM did not affect cathepsin A activity in melanoma cell line lysates. These data support our previous proposal that lactacystin is not a specific inhibitor of the proteasome. Since cathepsin A is also a tumor-associated enzyme, further research is needed to clarify its role and the significance of its inhibition by lactacystin in tumor biology.

Localization of a proteasomal antigen in human spermatozoa: immunohistochemical electron microscopic study.

The ultrastructural localization of a proteasomal antigen in human spermatozoa was studied by means of immunolabeling with the MPC21 monoclonal antibody and secondary gold labeled antibody with 1.4 nm gold particles in combination with silver enhancement reaction using pre-embedding technique. The labeling was found in the acrosomal and postacrosomal regions, in the connecting-piece (neck) and, in some cases, in the middle-piece and also in the residual bodies. There was no significant reaction in condensed chromatin. In some abnormal forms of spermatozoa, in which the chromatin was not well condensed, the labeling in nuclei was present. The nuclear vacuoles with looser chromatin were usually strongly labeled. The nuclei of cells representing different stages of spermatogenesis, that were present in semen samples, were also labeled.

Effects of an inhibitor of tripeptidyl peptidase II (Ala-Ala-Phe-chloromethylketone) and its combination with an inhibitor of the chymotrypsin-like activity of the proteasome (PSI) on apoptosis, cell cycle and proteasome activity in U937 cells.

AAF-AMC is not a specific TPP II substrate, since it is also hydrolyzed by purified proteasomes. Moreover, AAF-cmk, claimed to be a specific TPP II inhibitor, also inhibits the chymotrypsin-like activity of the proteasome. While AAF-cmk itself is mildly cytostatic to U-937 cells and induces cell cycle block in G1, its combination with PSI does not induce an increase in the cytostatic/cytotoxic effects. This suggests that TPP II is possibly less important for cell metabolism than it was previously believed and it is less probable that it can be able to fully compensate for the loss of the proteasome function.

Effects of proteasome inhibitor PSI on neoplastic and non-transformed cell lines.

In this study we compared the sensitivity of different human and murine cell lines varying in the stage of transformation to dose- and time-dependent cytostatic and/or cytotoxic effects of PSI (a selective proteasome inhibitor), measured by a standard MTT assay. It was found that intensively proliferating cell lines were more sensitive to very small doses of PSI after 24 h incubation than the slow proliferating ones. Non-transformed cell lines showed no sensitivity to PSI, as there was no difference in cell viability in comparison with the control group even after 72 h incubation.

Cyclodextrin enhances spermicidal effects of magainin-2-amide.

Magainins are antimicrobial peptides with known spermicidal activity. Their activity is inhibited by cholesterol present in eukaryotic cell membranes. Pretreatment of spermatozoa with methyl-beta-cyclodextrin, which extracts cholesterol from cell membranes and induces capacitation, sensitizes them to magainin-2-amide as shown by a decrease in human sperm motility determined by computer-assisted sperm analysis and a concomitant decrease in sperm viability, as measured by MitoTracker(R) Red CMXRos labeling. Magainin-2-amide affects mainly the fast progressive spermatozoa inducing them directly into an immotile state, without an increase in motile non-progressive and slow progressive spermatozoa. We conclude that methyl-beta-cyclodextrin highly potentiates the deleterious effect of magainin-2-amide on human spermatozoa. Most probably, this effect can be explained by cholesterol extraction from sperm cell membranes.

Lovastatin and simvastatin are modulators of the proteasome.

Lovastatin and simvastatin are HMG-CoA reductase inhibitors widely used as antihyperlipidemic drugs, which also display antiproliferative properties. In the present paper, we provide evidence that both lovastatin and simvastatin are modulators of the purified bovine pituitary 20 S proteasome, since they mildly stimulate the chymotrypsin-like activity and inhibit the peptidylglutamylpeptide hydrolyzing activity without interfering with the trypsin-like activity. However, those effects are only observed when the closed ring forms of the drugs are used, while the opened ring form of lovastatin acts as a mild inhibitor of the chymotrypsin like activity. The closed ring form of lovastatin is much more potent as a cytotoxic agent on the Colon-26 (C-26) colon carcinoma cell line than the opened ring form, which is only mildly cytostatic. Moreover, neither the cytotoxic effects nor the effects on 20 S proteasome activities are prevented by mevalonate, which by itself inhibits the trypsin-like activity of the proteasome. Neither the opened ring nor the closed ring form of lovastatin induces an accumulation of ubiquitin-protein conjugates, which is observed after treatment with lactacystin, a selective proteasome inhibitor. In contrast with the opened ring form of lovastatin, the closed ring form induces the disappearance of detectable p27(kip1) from C-26 cells. Altogether, our results indicate that the closed ring form of lovastatin induces cytotoxic effects independent of its HMG-CoA inhibiting activity, however, those effects are mediated by a complex modulation of proteasome activity rather than by inhibition of the 20 S proteasome.

Synergistic antitumor effects of a selective proteasome inhibitor and TNF in mice.

The ubiquitin-proteasome pathway is becoming an attractive target in cancer therapy. The inhibitors of proteasomes have recently been shown to induce apoptosis of tumor cells in vitro and to exert significant antitumor effects in murine tumor models in vivo. Proteasome inhibitors, also prevent NF-kappa B activation. Since this transcription factor is responsible for counteracting apoptosis induced by numerous agents, and proteasome inhibitors have already proved efficacious in increasing the proapoptotic activity of TNF in vitro, we decided to evaluate the antitumor effects of the combined PSI and TNF treatment against a murine C-26 carcinoma. Both agents separately exerted moderate antitumor efficacy. However, their combination proved to exert dramatic antitumor activity with retardation of tumor growth and prolongation of mice survival time. Moreover, 50% of the mice were completely cured by this drug combination. Unexpectedly, there was no potentiation of the cytostatic/cytotoxic effects of these drugs in in vitro assays which argues against the direct influence on C-26 cells. Similarly, the influence of these drugs on tumor induced angiogenesis does not seem to explain the observed antitumor effects. Further studies are necessary to explain the striking antitumor effects of the PSI and TNF combination.

Separation of cathepsin A-like enzyme and the proteasome: evidence that lactacystin/beta-lactone is not a specific inhibitor of the proteasome.

Previous studies have described a human platelet cathepsin A-like enzyme with a number of similarities to the "acidic" and "neutral" chymotrypsin-like activities of the proteasome. This includes its strong inhibition by the highly specific proteasome inhibitor Lactacystin/beta-lactone, suggesting that either the Cbz-Phe-Ala-hydrolyzing activity attributed to cathepsin A was due to the chymotrypsin-like activity of the proteasome or that lactacystin was not a specific inhibitor of the proteasome. In the present study we discard the first possibility on the basis of the following findings: (a) human platelet cathepsin A, unlike proteasome, binds to concanavalin A, and does not bind to Heparin-Sepharose at pH 7.4; (b) neither the chymotrypsin-like activity of the proteasome, nor proteasome antigens are detected in the cathepsin A preparation; (c) purified proteasome does not exhibit Cbz-Phe-Ala-hydrolyzing activity; (d) Z-lle-Glu-(Ot-Bu)Ala-leucinal (PSI), a compound that selectively inhibits the chymotrypsin-like activity of the proteasome at a concentration of 10 microM has no inhibitory effect on the carboxypeptidase activity of cathepsin A; (e) cathepsin A, free of the proteasome, is completely inhibited by micromolar concentrations of lactacystin/beta-lactone. It is therefore concluded that lactacystin/beta-lactone is not a specific inhibitor of the proteasome.

Proteasomes in human spermatozoa.

In the present study we describe the localization of proteasomes in human spermatozoa by means of immunolabelling with different monoclonal and polyclonal antibodies detected by confocal microscopy. Western blotting confirmed the specificity of the antibodies and has shown that proteasomes are present in spermatozoa and in seminal fluid. In spermatozoa proteasomes are concentrated in the neck region where the centrioles are located. Some labelling was also detected at the periphery of the head, but no proteasomal antigens were detected in either the nucleus or associated with the flagellum. Proteasome inhibitors did not affect the motility of the spermatozoa, acrosome reaction nor zona binding. It is hypothesized that paternal proteasomes enter the oocyte during fertilization in tight association with the centrioles and may serve a special function during further development which can be associated with the function of a hypothetical proteolysis centre.

Localization of proteasomes in human oocytes and preimplantation embryos.

In the present study we describe the localization of proteasomes in human oocytes, apoptotic preimplantation embryos, and triploid preimplantation embryos by means of immunolabelling with the MCP21 monoclonal antibody detected by confocal microscopy. While in the oocytes proteasomes are scattered throughout the cytoplasm, in the pronuclear zygote they appear to concentrate at the periphery of the cytoplasm and do not enter the pronuclei. During early cleavage stages, proteasome immunolabelling is concentrated in the nuclei, while the examination of triploid blastocysts showed that proteasomes had a similar cellular distribution to somatic cell lines, i.e. in the nuclei but not in the nucleoli or the cytoplasm. It appears that the distribution of proteasomes dramatically changes during human preimplantation embryo development.

Lovastatin induces mitotic abnormalities in various cell lines.

We examined the effects of lovastatin, a common anti-atherosclerotic drug and a blocker of the cell cycle, on the process of mitosis. It is known that lovastatin induces an arrest or a retardation of the cell cycle in many cell types not only at the G(1)phase, but also at the G(2)/M transition. After 24-48 h incubation of epithelial PtK(2), T24, HeLa cells and fibroblastic L929 cells in the presence of 1. 0-60.0 microm lovastatin, diverse mitotic perturbations have been observed. The most noteworthy phenomena recorded were prometaphase retardation and chromosome lagging during metaphase and anaphase. After the recovery in lovastatin-free media, the cells continued mitosis without any disturbances. Mevalonic acid prevented the effects of lovastatin. We conclude that the effects were specific for lovastatin-induced inhibition of mevalonic acid synthesis. Immunofluorescence studies with anticentromeric antibodies suggested that one of the possible causes of the lovastatin-induced mitotic disorder could be an interference with the development and function of the centromeres.