Thrombin generation assay using factor XIa to measure factors VIII and IX and their glycoPEGylated derivatives is robust and sensitive.

BACKGROUND: Conventional coagulation factor assays are associated with certain limitations, as they do not always reflect the clinical heterogeneity of bleeding in hemophilic patients or correctly reflect the individual patient response to treatment with bypassing agents or novel factor concentrates. The thrombin generation assay (TGA) is currently being assessed as a possible method for characterizing bleeding phenotypes in individuals with hemophilia. OBJECTIVES: This study assessed the robustness and sensitivity of the TGA for measuring the activity of recombinant factor VIII (rFVIII), recombinant factor IX (rFIX) and their glycoPEGylated derivatives, N8-GP and N9-GP, in vitro. METHODS: Factor-deficient plasma was spiked with 0.13-130 IU dL(-1) rFVIII or N8-GP (hemophilia A [HA] plasma), or rFIX or N9-GP (hemophilia B [HB] plasma). A calibrated automated thrombogram triggered with tissue factor (TF) or activated FXI (FXIa) was used to measure thrombin generation over time. Endogenous thrombin potential, peak thrombin, velocity index, lag time and time to peak thrombin were analyzed. RESULTS: FXIa-triggered assays were not affected by glycoPEGylation and were sufficiently sensitive to differentiate between spiked samples mimicking severe and moderate HB and HA; TF-triggered assays were not sufficiently sensitive for this distinction in HA. Both FXIa-triggered and TF-triggered assays had an acceptable level of variability (≤ 20%), although TF-triggered assays were associated with greater variability. CONCLUSIONS: FXIa-triggered TGA reactions produced more robust and sensitive results than TF-triggered TGA reactions, and have the potential for use in monitoring patients treated with glycoPEGylated or non-PEGylated coagulation factor concentrates. These promising results merit confirmation with clinical samples to correlate in vitro and in vivo data.

MCF-7/VD(R): a new vitamin D resistant cell line.

Several in vitro and in vivo experiments have demonstrated potent cell regulatory effects of vitamin D compounds in cancer cells. Moreover, a promising phase I study with the vitamin D analogue Seocalcitol (EB 1089) in patients with advanced breast and colon cancer has already been carried out and more clinical trials evaluating the clinical effectiveness of EB 1089 in other cancer types are in progress (Mørk Hansen et al. [2000a]). However, only little is known about the mechanisms underlying the actions of vitamin D or about the possible development of drug resistance in the patients. Therefore, in an attempt to gain more insight into these aspects, we have developed the MCF-7/VD(R) cell line, a stable subclone of the human MCF-7 breast cancer cell line, which is resistant to the growth inhibitory and apoptosis inducing effects of 1alpha,25(OH)(2)D(3). Despite this characteristic, receptor studies on the VDR have clearly demonstrated that the MCF-7/VD(R) cells contain fully functional VDRs, although in a lower number than seen with the parental MCF-7 cells. The regulation of the 24-hydroxylase enzyme appeared to be intact in the MCF-7/VD(R) cells and no differences with regard to growth rate and morphological appearance between the MCF-7/VD(R) cells and the parental MCF-7 cells were observed. Interestingly, however, the sensitivity of the MCF-7/VD(R) cells to the pure anti-estrogen ICI 182,780 was found to be increased. The MCF-7/VD(R) cell line shows characteristics different from those of previously described vitamin D resistant breast cancer cell lines but also some similarities. Together such vitamin D resistant cell lines therefore serve as a useful tool for studying the exact mechanism of action of vitamin D and the development of vitamin D resistance.

Vitamin D compounds exert anti-apoptotic effects in human osteosarcoma cells in vitro.

Several studies have demonstrated that vitamin D regulates growth and differentiation in bone cells in vitro. In addition, in vivo studies have shown that vitamin D stimulates bone formation, increases the number of osteoblast precursor cells and prevents bone mineral loss. These observations indicate that vitamin D may have anabolic effects on bone, and thus therapeutic potential in the treatment of osteoporosis. However, little is known about the effects of vitamin D on apoptosis in bone cells and about the contribution of this process to the effect of vitamin D on bone mineral loss. To investigate this aspect in more detail, we studied the effect of 1alpha,25(OH)(2)D(3) and a series of analogues on apoptosis in human osteosarcoma cells. No significant induction of apoptosis was observed with any of the compounds after a 5 day treatment period. In contrast, some of the analogues showed a tendency to protect the cells from undergoing apoptosis. This anti-apoptotic effect of vitamin D was further confirmed by the ability of 1alpha,25(OH)(2)D(3) to suppress camptothecin- and staurosporin-induced DNA fragmentation in the cells. In cultures treated simultaneously with 1alpha,25(OH)(2)D(3) in combination with camptothecin or staurosporin, the level of DNA fragmentation was markedly reduced compared with cultures treated with camptothecin or staurosporin alone. On the basis of the present results, it is therefore concluded that vitamin D displays anti-apoptotic effects in human osteoblast-like osteosarcoma cells in vitro. This observation suggests that besides regulating growth and differentiation, vitamin D exerts its anabolic effects on bone by protecting osteoblastic cells from undergoing apoptosis.

Cyanoguanidine CHS 828 induces programmed cell death with apoptotic features in human breast cancer cells in vitro.

The cyanoguanidine CHS 828 was recently shown to possess potent anti-tumour effects both in vitro and in vivo. The exact mechanism of action of CHS 828 is not known, but recent results have indicated that induction of programmed cell death may be one mechanism by which CHS 828 exerts its anti-tumour effects. To investigate this aspect in more detail, we studied the effect of CHS 828 and the reference compound Taxol beta on programmed cell death in human breast cancer cells in vitro. Both compounds were found to induce DNA fragmentation in the cells. However, microscopic examination of the cells demonstrated that CHS 828 and Taxol triggered different types of cell death. In the CHS 828-treated cultures most cells were found to be Annexin-V positive, indicating that these cells were early apoptotic cells, while no morphological characteristics of classical apoptosis were seen. In contrast, the cells in the Taxol-treated cultures displayed morphological features characteristic of classical apoptotic cells, but no Annexin-V positive cells could be observed. These findings together with the previously reported potent effects of CHS 828 on tumour cells, makes CHS 828 a promising new agent for the treatment of cancer patients.

Effect of calmodulin antagonists on endocytosis and intracellular transport of ricin in polarized MDCK cells.

The effect of calmodulin antagonists on endocytosis, transcytosis, recycling, and transport to the Golgi apparatus from both the apical and the basolateral plasma membrane of polarized Madin-Darby canine kidney cells has been investigated by using the plant toxin ricin as a membrane marker. The calmodulin antagonists trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) stimulated apical endocytosis of ricin, whereas basolateral endocytosis was unaffected. A stimulation of the apical uptake of the fluid-phase marker horseradish peroxidase by calmodulin antagonists was also found both by biochemical and by ultrastructural studies. Furthermore, W-7 reduced the recycling of ricin to the apical plasma membrane, whereas the recycling to the basolateral plasma membrane was not changed. Transport of ricin to the Golgi apparatus was also selectively affected by the calmodulin antagonist W-7. After basolateral endocytosis of ricin, transport to the Golgi apparatus was reduced, whereas after apical endocytosis the fraction of endocytosed ricin transport to the Golgi apparatus was increased. Transcytosis of ricin from the basolateral to the apical pole was increased in the presence of calmodulin antagonists, whereas these compounds did not have any significant effect on the apical to basolateral transcytosis. Thus, the results obtained indicate that calmodulin is involved in regulation of apical endocytosis and recycling as well as in transcytosis of ricin from the basolateral plasma membrane. Furthermore, the data suggest that calmodulin plays a role in regulation of ricin transport to the Golgi apparatus.

Destabilization of plasma membrane structure by prevention of actin polymerization. Microtubule-dependent tubulation of the plasma membrane.

Electron microscopy of thick (0.2-1.0 micron) sections of cytochalasin D-treated cells fixed in the presence of Ruthenium red revealed an extensive, surface-connected tubular compartment in HEp-2 cells. The tubules measured 120-220 nm in diameter and at least up to 6 microns in length. Morphometric analysis showed that in control cells about 0.2% of the total plasma membrane area (defined as all Ruthenium red-labeled membrane) appeared as vesicular or tubular profiles beneath the cell surface. However, after 15-30 minutes of cytochalasin D incubation about 4% of the total plasma membrane area is tubulated, and after 60-105 minutes as much as about 15% of the total plasma membrane appears as tubules. Clathrin-coated pits and caveolae-like structures were occasionally associated with the tubular membrane. Moreover, immunogold labeling showed that the tubular membrane contained transferrin receptors at about the same density as the nontubulated plasma membrane. Examination of cells in which endosomes and lysosomes were labeled with horseradish peroxidase before or after exposure to cytochalasin D showed that these organelles remained spherical, and that no horseradish peroxidase was present in the tubules. Moreover, the surface to volume ratio remained constant with increasing time of cytochalasin D incubation. Accordingly, the surface-connected tubules were not derived from endocytic structures but were formed by invagination of the plasma membrane. The tubule formation is reversible. When microtubules are depolymerized by nocodazole or colchicine treatment before the cells are exposed to cytochalasine D, tubule formation is strongly inhibited. Hence, the cytochalasin D-induced plasma membrane tubulation depends on intact microtubules.

Inhibition of the vacuolar H(+)-ATPase with bafilomycin reduces delivery of internalized molecules from mature multivesicular endosomes to lysosomes in HEp-2 cells.

In the human carcinoma cell line HEp-2, endosomes are multivesicular bodies (MVBs) which gradually mature and eventually fuse with other mature endosomes and/or with preexisting lysosomes. Selective inhibition of the vacuolar H(+)-ATPase with bafilomycin A1 (Baf) did not influence endocytic uptake and recycling of the protein toxin ricin. Moreover, quantification of immunogold labeling on ultracryosections revealed that the frequency by which MVBs containing internalized cationized gold (Cat.Au) also labeled for the cation-independent mannose-phosphate receptor (MPR) was the same with and without Baf, suggesting that formation and maturation of MVBs were unchanged in the presence of Baf. However, degradation of ricin was strongly reduced by bafilomycin, and this reduction was not only due to increased pH in lysosomes. Thus, quantitative lmmunogold labeling showed that the Baf-induced alkalinization reduced the transfer of internalized Cat.Au to lysosomes, as defined by their content of human lysosome-associated membrane protein 1 (h-lamp-1) and cathepsin D. Accordingly, although low vacuolar pH does not seem to be required for transport to MPR-containing endosomes, it seems to be important for a late fusion step along the endocytic pathway.

Role of processing and intracellular transport for optimal toxicity of Shiga toxin and toxin mutants.

Cleavage of Shiga toxin A-fragment at a highly trypsin-sensitive site increases its enzymatic activity. To investigate the role of this cleavage site in intoxication of cells, we studied the routing, cleavage, and toxicity of mutant toxin where the trypsin-sensitive site had been eliminated. Ultrastructural analysis of toxin tagged with horseradish peroxidase demonstrated that wild-type and mutant toxins were transported from endosomes to the trans-Golgi network and further through the Golgi cisterns to the endoplasmic reticulum. Wild-type toxin was much more efficient than the mutants in provoking rapid intoxication, but after prolonged incubation time also mutants were highly toxic. The cells were able to cleave both wild-type Shiga toxin and the mutants, but the cellular location for cleavage appears to differ. Wild-type toxin was cleaved in the presence of brefeldin A, which disrupts the Golgi cisterns. This indicates that the cleavage occurs in the endosomes or in the trans-Golgi network. In contrast, the mutant Shiga-His (R248H/R251H) was not cleaved in the presence of brefeldin A, indicating that the cleavage can occur only after the toxin has left the trans-Golgi network. In vitro experiments showed that the cytosolic enzyme calpain is able to cleave Shiga-His, and results from in vivo experiments are consistent with the possibility that cleavage is carried out by calpain after the mutant A-fragment has reached the cytosol.

Delivery to lysosomes in the human carcinoma cell line HEp-2 involves an actin filament-facilitated fusion between mature endosomes and preexisting lysosomes.

We have addressed the following question: what is the mechanism behind the delivery of internalized molecules from mature endosomes to lysosomes in HEp-2 cells, and which role does the cytoskeleton play in this process? Quantitative electron microscopy and immunogold labeling revealed that whereas the cytoskeleton was not of importance for endosome maturation, actin filaments facilitated fusion of mature endosomes with preexisting lysosomes. Delivery to lysosomal degradation was not dependent on protein synthesis as determined biochemically, but was reduced by cytochalasin D. Observations made by electron microscopy as well as by video microscopy of living cells showed that the concerted action of actin filaments and microtubules was responsible for the random distribution and movement of endocytic organelles throughout the cell. Actin microfilaments, however, seem to facilitate perinuclear clustering and frequent fusion of mature endosomes and lysosomes, while microtubules play a role in preventing formation of large lysosome aggregates by separating endosomes and lysosomes and moving them toward the cell periphery. Taken together, our data suggest that delivery of internalized molecules to lysosomal proteolysis takes place by fusion of mature endosomes with preexisting lysosomes and that actin microfilaments somehow facilitate this step.

Phorbol myristate acetate selectively stimulates apical endocytosis via protein kinase C in polarized MDCK cells.

We have examined the regulation of endocytosis in polarized Madin-Darby canine kidney (MDCK) cells. Using quantitative electron microscopy and biochemical measurements, we found that incubation with the tumor promoter phorbol 12-myristate 13-acetate (TPA) stimulated endocytosis of horseradish peroxidase (HRP) and ricin four- to fivefold at the apical side in MDCK cells, whereas the uptake at the basolateral membrane was unaffected. The use of several protein kinase inhibitors and TPA analogues indicated that the stimulation of apical endocytosis was mediated via protein kinase C independently of protein kinase A. This stimulation occurred even when the clathrin-dependent pathway was inhibited by acidification of the cytosol, suggesting that the TPA-stimulated uptake was associated with a clathrin-independent mechanism. Moreover, we found that TPA also stimulated recycling of ricin to the apical domain. Ultrastructural analysis of MDCK cells preincubated with TPA revealed that neither the morphology nor the size of the endosomes was altered compared to control cells. Using morphometry, no marked change in the apical plasma membrane area was detected after incubation with TPA. These data indicate that the TPA-stimulated endocytosis involved neither ruffling nor formation of macropinosomes in MDCK cells. Finally, we found that TPA also selectively stimulated apical endocytosis in the human colon adenocarcinoma cell line (Caco-2). The data suggest that protein kinase C is involved in a strong stimulation of apical endocytosis and might participate in the regulation of membrane trafficking between the apical plasma membrane and apical endosomes in polarized epithelial cells.

Retrograde transport from the Golgi complex to the ER of both Shiga toxin and the nontoxic Shiga B-fragment is regulated by butyric acid and cAMP.

Endocytosed Shiga toxin is transported from the Golgi complex to the endoplasmic reticulum in butyric acid-treated A431 cells. We here examine the extent of this retrograde transport and its regulation. The short B fragment of Shiga toxin is sufficient for transport to the ER. The B fragment of cholera toxin, which also binds to glycolipids, is transported to all the Golgi cisterns, but cannot be localized in the ER even after butyric acid treatment. Under all conditions the toxic protein ricin was found predominantly in the trans-Golgi network. There is no transport of endocytosed fluid to the Golgi apparatus or to the ER even after butyric acid treatment and in the presence of Shiga toxin, indicating that transport to the ER, through the trans-Golgi network and the cisterns of the Golgi apparatus, involves several sorting stations. Since Shiga toxin receptors (Gb3) in butyric acid-treated A431 cells seem to have a ceramide moiety with longer fatty acids than in untreated cells, the possibility exists that fatty acid composition of the receptor is important for sorting to the ER. Both retrograde transport and intoxication with Shiga toxin can also be induced by cAMP, supporting the idea that retrograde transport from the Golgi to the ER is required for intoxication. The data suggest that transport to the ER in cells in situ may depend on fatty acid composition and is regulated by physiological signals.

Selective regulation of apical endocytosis in polarized Madin-Darby canine kidney cells by mastoparan and cAMP.

We here demonstrate that mastoparan, fluoride, forskolin, cholera toxin, and 8-bromoadenosine-3'-5'-cyclic monophosphate all selectively stimulate apical endocytosis of the protein toxin ricin without increasing the uptake at the basolateral side of polarized Madin-Darby canine kidney cells. Activation of adenylyl cyclase and an increased level of cAMP seem to increase ricin endocytosis by a clathrin-independent mechanism since stimulation of endocytosis by cholera toxin and 8-bromoadenosine-3'-5'-cyclic monophosphate occurred even when the clathrin-dependent pathway was blocked by low cytosolic pH. The data suggest that mastoparan stimulates apical endocytosis by interacting with heterotrimeric G proteins, and also this stimulation of endocytosis appears to be clathrin independent since the uptake of transferrin at the apical side was strongly inhibited by mastoparan after brefeldin A-induced missorting of the transferrin receptor to this pole of the cell. In addition, mastoparan stimulated apical endocytosis when clathrin-mediated endocytosis was blocked by acidification of the cytosol. Furthermore, we provide evidence for the existence of clathrin-independent endocytosis on both the apical and the basolateral surface of control Madin-Darby canine kidney cells. Our results suggest that endocytosis at the apical pole of epithelial cells can be regulated selectively by a physiological signal.

Endocytosis of desmosomal plaques depends on intact actin filaments and leads to a nondegradative compartment.

Epithelial cells in situ can internalize their desmosomes. This can be induced in cell cultures after removal of calcium ions from the cell medium. To study this endocytic process, a nontumorigenic human breast epithelial cell line, HMT-3522, was used. HMT-3522 cells were grown in serum-free, chemically defined medium, containing epidermal growth factor (EGF). Removal of EGF from the medium led to growth arrest and a kind of epithelial differentiation process in which adjacent cells interdigitated and formed more desmosomes than in the proliferating state. Growth-inhibited HMT-3522 cells dissociated following EGTA treatment, the desmosomes divided in a symmetrical fashion, and the desmosomal plaques (half-desmosomes) on the cell surface became internalized. The internalization was independent of clathrin, since immunogold labeling of ultracryosections never showed clathrin on desmosomal plaque-associated membrane domains. Moreover, cytosol acidification, which selectively inhibits endocytosis from clathrin-coated pits, practically blocked the uptake of transferrin, whereas internalization of desmosomal plaques continued. In contrast, actin filaments appeared to be involved in the desmosomal internalization. Thus, depolymerization of actin filaments by cytochalasin D significantly reduced endocytosis of half-desmosomes. Immunogold labeling showed that the vesicles with desmosomal plaques were not enriched in MPR (cation-independent mannose-6-phosphate receptor), cathepsin D or the lysosome-associated membrane protein lamp-1. In addition, the morphology was different. Thus, the endocytic vesicles with desmosomal plaques represent a special compartment, distinct from typical endosomes and lysosomes.

Multivesicular bodies in HEp-2 cells are maturing endosomes.

Conventional fluorescence microscopy of fixed HEp-2 cells as well as video microscopy of living cells incubated with transferrin-Texas Red (Tf-TxR) for < 60 min revealed distinct punctuate endosomal structures. Quantitative ultrastructural analysis using horseradish peroxidase (HRP) and cationized gold as tracers showed that spherical multivesicular bodies (MVBs) were the predominant endocytic compartments in HEp-2 cells and that MVBs within 60 to 90 min matured into lysosomes still containing internal vesicles. The number of labeled MVBs increased continuously from 2.5 min to 30 min of tracer incubation. However, when the cells were pulsed for 5 min followed by 10 or 25 min chases, the number of labeled MVBs corresponded to that obtained after 5 min of continuous incubation. The diameter of labeled MVBs was largely constant with time, but the number of internal MVB vesicles increased. Thus, early or newly formed MVBs contained few internal vesicles, whereas late MVBs, that is to say, MVBs that have existed for some period of time, contained numerous internal vesicles, and finally a mixture of membranous material or myelin figures and vesicles. It is thus in principle possible to distinguish between early and late MVBs in HEp-2 cells on the basis of morphology. However, the difference in number of internal vesicles applies only to the entire MVB population; after only 2.5 to 5 min of incubation, MVBs with numerous internal vesicles could also be reached by internalized tracer. Concomitant with the gradual changes in morphology, the MVBs also showed a characteristic change in content of marker proteins as detected by immunogold labeling on ultracryosections. Hence, early MVBs with relatively few internal vesicles and typically reached by internalized tracers within 5 min contained transferrin receptors (TfRs). By contrast, MVBs with many internal vesicles and labeled after 60 min of incubation contained mannose-phosphate receptors (MPRs), and the MVBs with distinct membranous material or myelin figures in addition to the internal vesicles were enriched in the lysosome membrane protein lamp-1. Thus, there seems to be a gradual maturation of MVBs in HEp-2 cells.

Are caveolae involved in clathrin-independent endocytosis?

In addition to endocytosing molecules via clathrin-coated pits, cells also internalize membrane and fluid by a clathrin-independent endocytic mechanism. In this article we search for the equivalent of clathrin-coated pits in clathrin-independent endocytosis, and discuss some pitfalls in the interpretation of electron micrographs. We also discuss how the early steps in clathrin-independent endocytosis might be analysed morphologically, and we argue that caveolae are not involved in clathrin-independent endocytosis.