Occurrence of lysosomes and the differentiation of rat liver cells

Summary The occurrence of lysosomes has been investigated electron microscopically and cytochemically in cells of rat liver in the course of ontogenesis.It has been found that primary lysosomes occur during the whole period under investigation and that they originate from the Golgi complex. Some of them assume the appearance of multivesicular bodies. Acid phosphatase activity is lower at the prenatal stage than after the birth. The occurrence of secondary lysosomes proceeds in two stages. Secondary lysosomes appear in a high number at the beginning of differentiation of the liver diverticulum (10–12 day of embryonic life). On the subsequent days they are, with few exceptions, no more present. At the end of the embryonic period (starting with the 20th day) and especially after the birth, they progressively grow in number and move from the region of central cytoplasm peripherally towards the bile capillary.Differences in occurrence of secondary lysosomes are in connexion with reconstruction of the liver primordium at the beginning of liver development and with the change in metabolism of the liver cell after the birth.     

A novel kinesin-like protein, KIF1Bbeta3 is involved in the movement of lysosomes to the cell periphery in non-neuronal cells

The kinesin superfamily protein, KIF1Bβ, a splice variant of KIF1B, is involved in the transport of synaptic vesicles in neuronal cells, and is also expressed in various non-neuronal tissues. To elucidate the functions of KIF1Bβ in non-neuronal cells, we analyzed the intracellular localization of KIF1Bβ and characterized its isoform expression profile. In COS-7 cells, KIF1B colocalized with lysosomal markers and expression of a mutant form of KIF1Bβ, lacking the motor domain, impaired the intracellular distribution of lysosomes. A novel isoform of the kinesin-like protein, KIF1Bβ3, was identified in rat and simian kidney. It lacks the 5th exon of the KIF1Bβ-specific tail region. Overexpression of KIF1Bβ3 induced the translocation of lysosomes to the cell periphery. However, overexpression of KIF1Bβ3-Q98L, which harbors a pathogenic mutation associated with a familial neuropathy, Charcot-Marie-Tooth disease type 2 A, resulted in the abnormal perinuclear clustering of lysosomes. These results indicate that KIF1Bβ3 is involved in the translocation of lysosomes from perinuclear regions to the cell periphery.     

A novel method of imaging lysosomes in living human mammary epithelial cells

Cancer cells invade by secreting degradative enzymes which, under normal conditions, are sequestered in lysosomal vesicles. The ability to noninvasively label lysosomes and track lysosomal trafficking would be extremely useful to understand the mechanisms by which degradative enzymes are secreted in the presence of pathophysiological environments, such as hypoxia and acidic extracellular pH, which are frequently encountered in solid tumors. In this study, a novel method of introducing a fluorescent label into lysosomes of human mammary epithelial cells (HMECs) was evaluated. Highly glycosylated lysosomal membrane proteins were labeled with a newly synthesized compound, 5-dimethylamino-naphthalene-1-sulfonic acid 5-amino-3,4,6-trihydroxy-tetrahydro-pyran-2-ylmethyl ester (6-O-dansyl-GlcNH2). The ability to optically image lysosomes using this new probe was validated by determining the colocalization of the fluorescence from the dansyl group with immunofluorescent staining of two well-established lysosomal marker proteins, LAMP-1 and LAMP-2. The location of the dansyl group in lysosomes was also verified by using an anti-dansyl antibody in Western blots of lysosomes isolated using isopycnic density gradient centrifugation. This novel method of labeling lysosomes biosynthetically was used to image lysosomes in living HMECs perfused in a microscopy-compatible cell perfusion system.     

A synthetic tripeptide which increases survival of normal liver cells, and stimulates growth in hepatoma cells

A synthetic peptide with a structure analogous to a growth promoting human serum tripeptide was found to possess activities which, at nanomolar concentrations, increased the survival of normal hepatocytes from regenerating rat liver and which enhanced growth of a line of cultured hepatoma cells. The synthetic tripeptide (glycyl-histidyl-lysine) also stimulated the incorporation of labeled uridine and thymidine into trichloroacetic acid precipitable material.     

A novel protocol for the subcellular fractionation of C3A hepatoma cells using sucrose density gradient centrifugation

In this paper, we describe a method to obtain a relatively pure mitochondrial and microsomal fractions by subcellular fractionation of human hepatoma cell line C3A using sucrose as the hypoosmotic medium. The cells were subjected to osmotic stress with sucrose and homogenized. Osmolarity was then restored to the cells and the organelles were separated by density gradient centrifugation. The protein profiles were examined by SDS-PAGE and the purity was analysed by marker enzymes and Western blotting. Our results indicate a good separation of mitochondrial and microsomal fractions from human hepatoma C3A cells.     

A bifunctional allosteric site in the dimer interface of procaspase-3

The dimer interface of caspase-3 contains a bifunctional allosteric site in which the enzyme can be activated or inactivated, depending on the context of the protein. In the mature caspase-3, the binding of allosteric inhibitors to the interface results in an order-to-disorder transition in the active site loops. In procaspase-3, by contrast, the binding of allosteric activators to the interface results in a disorder-to-order transition in the active site. We have utilized the allosteric site to identify a small molecule activator of procaspase and to characterize its binding to the protease. The data suggest that an efficient activator must stabilize the active conformer of the zymogen by expelling the intersubunit linker from the interface, and it must interact with active site residues found in the allosteric site. Small molecule activators that fulfill the two requirements should provide scaffolds for drug candidates as a therapeutic strategy for directly promoting procaspase-3 activation in cancer cells.     

Photofrin binds to procaspase-3 and mediates photodynamic treatment-triggered methionine oxidation and inactivation of procaspase-3

Diverse death phenotypes of cancer cells can be induced by Photofrin-mediated photodynamic therapy (PDT), which has a decisive role in eliciting a tumor-specific immunity for long-term tumor control. However, the mechanism(s) underlying this diversity remain elusive. Caspase-3 is a critical factor in determining cell death phenotypes in many physiological settings. Here, we report that Photofrin-PDT can modify and inactivate procaspase-3 in cancer cells. In cells exposed to an external apoptotic trigger, high-dose Photofrin-PDT pretreatment blocked the proteolytic activation of procaspase-3 by its upstream caspase. We generated and purified recombinant procaspase-3-D₃A (a mutant without autolysis/autoactivation activity) to explore the underlying mechanism(s). Photofrin could bind directly to procaspase-3-D₃A, and Photofrin-PDT-triggered inactivation and modification of procaspase-3-D₃A was seen in vitro. Mass spectrometry-based quantitative analysis for post-translational modifications using both ¹⁶O/¹⁸O- and ¹⁴N/¹⁵N-labeling strategies revealed that Photofrin-PDT triggered a significant oxidation of procaspase-3-D₃A (mainly on Met-27, -39 and -44) in a Photofrin dose-dependent manner, whereas the active site Cys-163 remained largely unmodified. Site-directed mutagenesis experiments further showed that Met-44 has an important role in procaspase-3 activation. Collectively, our results reveal that Met oxidation is a novel mechanism for the Photofrin-PDT-mediated inactivation of procaspase-3, potentially explaining at least some of the complicated cell death phenotypes triggered by PDT.     

A role for small GTPase RhoA in regulating intracellular membrane traffic of lysosomes in invasive rat hepatoma cells

Small GTPase RhoA regulates signal transduction from receptors in the membrane to a variety of cellular events related to cell morphology, motility, cytoskeletal dynamics, cytokinesis, and tumour progression, but it is unclear how RhoA regulates intracellular membrane dynamics of lysosomes. We showed previously by confocal immunofluorescence microscopy that the transfection of dominant active RhoA in MM1 cells causes the dispersal translocation of lysosomes stained for cathepsin D throughout the cytoplasm. Y-27632, a selective inhibitor of p160ROCK, impeded the cellular redistribution of lysosomes and promoted reclustering of lysosomes toward the perinuclear region. Here we have further investigated whether the acidic lysosomal vesicles dispersed throughout the cytoplasm are applied to the early endosomes in the endocytic pathway, and we demonstrate that the dispersed lysosomes were accessible to endocytosed molecule such as dextran, and their acidity was not changed, as determined by increased accumulation of the acidotropic probe LysoTracker Red. Brefeldin A did not induce the tabulation of these dispersed lysosomes, but it caused early endosomes to form an extensive tubular network. The dispersed lysosomes associated with cathepsin D and LIMPII were not colocalized with early endosomes, and these vesicles were not inaccessible to the endocytosed anti-transferrin receptor antibody. Moreover, wortmannin, an inhibitor of phosphatidylinositol 3-kinase, induced a dramatic change in LIMPII-containing structures in which LIMPII-positive swollen large vacuoles were increased and small punctate structures disappeared in the cytoplasm. These swollen vacuoles were not doubly positive for LIMPII and transferrin receptor, and were not inaccessible to the internalized anti-transferrin receptor antibody. Therefore, our novel findings presented in this paper indicate that RhoA activity causes a selective translocation of lysosomes without perturbing the machinery of endocytic pathway.     

Fine structure and cytochemistry of lysosomes in the Ito cells of the rat liver

Summary Ultracytochemical studies of the performic acid-phosphotungstic acid (PFP) reaction and acid phosphatase (ACPase) activity in the Ito cells (fat-storing cells) of the rat liver revealed two kinds of lipid droplets: one surrounded by a structure giving PFP- and ACPase-positive reactions, recognized as a lysosome, the other without such a reactive structure displaying a limiting membrane.To elucidate the function of the lysosomes surrounding lipid droplets, experiments were carried out on the following groups of animals: (1) Vitamin A-deficient rats were fed a normal diet containing vitamin A, and (2) hypervitaminosis A was experimentally induced in previously untreated rats. Lipid droplets were studied in both groups.No lipid droplets reappearing in an early stage after restoration of the regular diet were either membrane-bounded or surrounded by lysosomes. Lipid droplets surrounded by lysosomes could be seen in rats fully restored from vitamin-A deficiency and more frequently in animals suffering from hypervitaminosis A. It seems likely that as a result of the lysosomal activity in the immediate vicinity of the lipid droplets a degradation of the vitamin A-containing lipid droplets takes place in the Ito cells. Therefore, the lysosome-surrounded lipid droplets can be regarded as a sort of autophagolysosome; these lysosomes may play a role in preventing an unrestricted increase in the number and volume of lipid droplets.This work was supported by Grants-in-Aid for Co-operative Research (Nos. 437001 and 57370001) from the Ministry of Education, Science and Culture, Japanese Government     

Inhibition of the discharge of endocytosed protein from phagosomes into lysosomes in hepatoma cells exposed to dimerized ribonuclease A.

The mechanism of the cytostatic action of dimerized ribonuclease A toward cultured hepatoma cells was investigated. A decrease in mitotic index, modifications of adsorptive properties of the pericellular membrane and inhibition of the degradation of two different proteins taken up by endocytosis are the first cell functions to be affected by the dimer. This effect on protein digestion is not due to an inhibition of proteolytic enzymes. The intracellular localization of exogenous protein and of ribonuclease dimer was studied by cell fractionation. When proteins (horseradish peroxidase or rabbit immunoglobulin G) are taken up by control hepatoma cells, they are first associated with phagosomes equilibrating at a lower density than lysosomes; their density distribution gradually becomes similar to that of lysosomes. When cells are pre-exposed to ribonuclease dimer, this modification of the density distribution as a function of time no longer occurs, although these proteins are still intracellular, as indicated by fractionation by differential centrifugation. During the first hour after addition of ribonuclease dimer, kinetic studies show an increased fixation of peroxidase to the cell membrane. Protein release into the culture medium is also increased. These results can be explained either by an absence of fusion between phagosomes and lysosomes, or by an inhibition of the discharge of peroxidase adsorbed to the phagosomal membrane after fusion.     

Kinetic comparison of procaspase-3 and caspase-3

Caspases, the key enzymes in apoptosis, are synthesized as proenzymes and converted into active form by proteolytic cleavage. The residues on active site reorganize during the activation process as shown in the comparative studies of crystallographic structures of procaspase-7 and its mature form. On the other hand, the proenzyme itself has some activity. Aiming to characterize the activation process, the comparative kinetic study for the pro- and mature caspase-3 was performed. In 1/K(M) versus pH study, a residue with pKa of 6.89+/-0.13 was detected only in caspase-3. While Vmax versus pH kinetic results were consistent with the existence of a residue with pKa of 6.21+/-0.06 in procaspase-3 mutant (D9A/D28A/D175A) but not in caspase-3. In the inactivation assays with diethylpyrocarbonate, a residue (pKa, 6.61+/-0.05) could be determined only for caspase-3 whereas with iodoacetamide a residue with pKa value (6.01+/-0.05) could be assigned only for procaspase-3. Considering that those residues could be protected by caspase-3-specific inhibitor from the inactivation, the modifiers are histidine- and cysteine-specific, respectively, and the involvement of these residues in the characteristic catalytic dyad of caspases, the results indicate that the pKa values of the catalytic histidine and cysteine residues are changed during the activation process.     

Effect of amines on the pH in the lysosomes of 3T3 cells

The measurements of intralysosomal pH under the action of the number of amines earlier reported to block the process of the initiation of cell proliferation (Nikolsky et al., 1984) were made on Swiss 3T3 cells. The intralysosomal pH (pH1) value was estimated by parameters of fluorescence of fluorescein-labeled dextran in single intact cells. The pHl value was equal to 4.7 +/- 0.2 for both actively growing and quiescent cells. The pH gradient between lysosomes and the cytoplasm was completely destroyed by monensin and partially by carbonylcyamide-m-chlorophenylhydrasone. Methylamine and chloroquine rapidly enhanced the pHl, value to 6.4-7.0. Dansylcadaverine, 5-methoxytryptamine and dimethylurea did not affect pHl value. Intracellular accumulation of dansylcadaverine was shown to be due to the existence of acidic compartments into the cell and highly decreased in the presence of monensin. A conclusion is made that the inhibition of mitogenic signal by amines cannot be unequivocally accounted for by increasing the pH in organelles involved in the intracellular processing of growth factors.     

Patt1, a novel protein acetyltransferase that is highly expressed in liver and downregulated in hepatocellular carcinoma,enhances apoptosis of hepatoma cells

Protein acetylation is increasingly recognized as an important post-translational modification. Although a lot of protein acetyltransferases have been identified, a few putative acetyltransferases are yet to be studied. In this study, we identified a novel protein acetyltransferase, Patt1, which belongs to GNAT family. Patt1 exhibited histone acetyltransferase activity and auto-acetylation activity. Deletion and mutation analysis of the predicted acetyltransferase domain in Patt1 showed that the conserved Glu139 was an important residue for its protein acetyltransferase activity. Furthermore, we found that Patt1 was highly expressed in liver and significantly downregulated in hepatocellular carcinoma tissues. In addition, we showed that overexpression of Patt1 enhanced the apoptosis of hepatoma cells dependent on its acetyltransferase activity, whereas knockdown of Patt1 significantly protected Chang liver cells from apoptosis. These data suggest that Patt1 might be involved in the development of hepatocellular carcinoma, and could be served as a potential therapy target for hepatocellular carcinoma.