BBA - Molecular Cell Research (v.1541, #3)

Internalization and stepwise degradation of heparan sulfate proteoglycans in rat hepatocytes by Morten Egeberg; Rune Kjeken; Svein O. Kolset; Trond Berg; Kristian Prydz (135-149).
Intracellular transport and degradation of membrane anchored heparan sulfate proteoglycans (HSPGs) were studied in cultured rat hepatocytes labeled with [35S]sulfate and [3H]glucosamine. Pulse chase experiments showed that membrane anchored HSPGs were constitutively transported to the cell surface after completion of polymerization and modification of the glycosaminoglycan chains in the Golgi apparatus. The intact HSPGs had a relatively short residence time at the cell surface and in non-degrading compartments (T 1/2 ∼2–3 h), while [35S]sulfate labeled degradation products were found in lysosomes, and to a lesser extent in late endosomes. These degradation products which were free heparan sulfate chains with little or no protein covalently attached, were approximately half the size of the original glycosaminoglycan chains and were the only degradation intermediate found in the course of HSPG catabolism in these cells. In cells incubated in the presence of the microtubule perturbant vinblastine, or in the presence of the vacuolar ATPase inhibitor bafilomycin A1, and in cells incubated at 19°C, the endocytosed HSPGs were retained in endosomes and no degradation products were detected. Disruption of lysosomes with glycyl-phenylalanine 2-naphthylamide (GPN) revealed a GPN resistant degradative compartment with both intact and partially degraded HSPGs. This compartment probably corresponds to late endosomes. Treatment of hepatocytes with the thiol protease inhibitor leupeptin inhibited the final degradation of the protein moiety of the HSPGs. The protein portion seems to be degraded completely before the glycosaminoglycan chains are cleaved. The degradation of the glycosaminoglycan chains is rapid and complete with one observable intermediate.
Keywords: Heparan sulfate proteoglycan; Hepatocyte; Bafilomycin; Endocytosis; Leupeptin; Lysosome; Vinblastine;

Pharmacological in vitro evaluation of new substance P–cyclodextrin derivatives designed to drug targeting towards NK1-receptor bearing cells by Christophe Péan; Anne Wijkhuisen; Florence Djedaïni-Pilard; Jacqueline Fischer; Stéphane Doly; Marie Conrath; Jean-Yves Couraud; Jacques Grassi; Bruno Perly; Christophe Créminon (150-160).
Some biological properties of new bifunctional conjugates designed for drug targeting were evaluated through in vitro experiments. Eight peptidylcyclodextrin compounds were used, which correspond to modified β- or γ-cyclodextrin (CD) grafted on neuropeptide substance P (SP) or a shorter derivative (SP(4–11)). Using anti-SP and anti-CD antibodies as molecular probes, we showed that the main structural features of the two moieties of these adducts were preserved. Binding experiments, using CHO cells expressing the human SP-specific NK1 receptor, demonstrated the functionality of all peptidylcyclodextrin derivatives, which exhibited IC50 values in a 10−9–10−7 M range. All compounds were able to induce a pharmacological response, triggering phosphatidylinositol turnover with EC50 values in the same range as the natural ligand. Moreover, autoradiography analysis of rat spinal corn sections proved that [125I]SP binding was dose-dependently displaced by one selected compound (a γ-CD–SP), showing a similar affinity of this adduct for the rat neurokinin 1 receptor. Our observations demonstrate that these peptidylcyclodextrins efficiently target NK1 receptor-expressing cells.
Keywords: Cyclodextrin; Substance P; Neurokinin 1 receptor; Tachykinin; Supramolecular chemistry; Drug targeting;

Signal transduction through cAMP to activate gene expression via the cAMP-responsive element (CRE) is one of the most intensively studied transcription pathways. In this pathway, transcription factor CRE-binding protein (CREB) recognizes the CRE enhancer on DNA. The CREB protein is activated via phosphorylation at serine 133 by protein kinase A and then is able to recruit coactivator CREB-binding protein (CBP) and its homologue p300. This recruitment of CBP/p300 is required for transcription activation. The mechanism for CBP/p300 to participate in this transcription process is still unclear. CBP and p300 are histone acetyltransferases (HAT) and able to associate with other HAT proteins. It has been reported that the regulation of nuclear receptor-mediated transcription initiation by p300 requires chromatin and its HAT function. The data shown here indicate that the requirements for chromatin and p300 HAT activity also apply to the activation of CREB-mediated transcription. Serine 133-phosphorylated CREB recruits p300 onto chromatin for efficient acetylation of nucleosomes. This targeted acetylation by p300 is essential to CREB-dependent transcription pathway.
Keywords: Chromatin; cAMP-responsive element binding protein; Histone acetyltransferase; p300; Transcription;

In a recent work [Photochem. Photobiol. B: Biol. 50 (1999) 8] the successful photodynamic inactivation of Escherichia coli bacteria by visible light was reported based on δ-aminolevulinic acid (ALA)-induced endogenous porphyrin accumulation. In this work, the identification of these porphyrin derivatives in intact bacteria was performed by low-temperature conventional fluorescence and fluorescence line narrowing (FLN) techniques. Conventional fluorescence emission spectroscopy at cryogenic temperatures revealed the presence of the free-base porphyrins, identified earlier by high-performance liquid chromatography analysis of disintegrated bacterial cells after ALA induction; however, emission maxima characteristic for metal porphyrins were also observed. We demonstrated that the primary reason for this signal is that metal porphyrins are formed from free-base porphyrins by Mg2+ ions present in the culturing medium. Incorporation of Zn ions originating from the glassware could also be supposed. In the FLN experiment, the energy selection effect could be clearly demonstrated for (0,0) emissions of both the free-base and the metal porphyrins. The comparison of the conventional emission spectra and the bands revealed by the FLN experiment show that the dominant monomeric structural population is that of metal porphyrins. The intensity and the shape of the FLN lines indicate an aggregated population of the free-base porphyrins, beside a small monomeric population.
Keywords: Photodynamic inactivation; Bacteria; Fluorescence line narrowing; Inhomogeneous distribution function; Fluorescence spectroscopy; Escherichia coli;

The tyrosine kinase c-Src is required for 1,25(OH)2-vitamin D3 signalling to the nucleus in muscle cells by Claudia Buitrago; Ricardo Boland; Ana Russo de Boland (179-187).
We have recently shown that the hormonal form of vitamin D3, 1,25(OH)2-vitamin D3 (1,25(OH)2D3), stimulates the enzymatic activity of the non-receptor protein tyrosine kinase c-Src in skeletal muscle cells. In this study we show that intracellular and extracellular Ca2+ chelation with BAPTA and EGTA, respectively, blocked hormone stimulation of c-Src activity/dephosphorylation, indicating that the calcium messenger system is an upstream activator of c-Src. Tyrosine phosphorylation and stimulation of the growth-related mitogen-activated protein kinase (MAPK) by 1,25(OH)2D3 was shown to be dependent on activation of c-Src, since pretreatment with the c-Src specific inhibitor PP1 or muscle cell transfection with an antisense oligodeoxynucleotide directed against c-Src mRNA markedly reduced hormone stimulation of MAPK phosphorylation. Evidence was obtained indicating that MAPK is then translocated to the cell nucleus in active phosphorylated form and induces the expression of c-myc oncoprotein, as the MAPK kinase (MEK) inhibitor PD98059 abolished stimulation of c-myc synthesis by 1,25(OH)2D3. In addition, the hormone rapidly stimulated tyrosine phosphorylation of c-myc. In cells pretreated with PP1 (4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo-D3,4-pyrimidine), the 1,25(OH)2D3-induced increase in tyrosine phosphorylation of c-myc was suppressed. Taken together, these results demonstrate that 1,25(OH)2D3 stimulates proliferation-associated signalling pathways in skeletal muscle cells and implicate c-Src kinase as mediator of this response.
Keywords: 1,25(OH)2-vitamin D3; Tyrosine phosphorylation; c-Src kinase; MAPK; Skeletal muscle cells; Signal transduction;

Prevention of docosahexaenoic acid-induced cytotoxicity by phosphatidic acid in Jurkat leukemic cells: the role of protein phosphatase-1 by Rafat A. Siddiqui; Laura J. Jenski; Jacqueline D. Wiesehan; Michelle V. Hunter; Richard J. Kovacs; William Stillwell (188-200).
The present investigation explores the role of phosphatidic acid (PA), a specific protein phosphatase-1 (PP1) inhibitor, in cytotoxicity induced by docosahexaenoic acid (DHA). The cytotoxicity of DHA was assayed by quantifying cell survival using the trypan blue exclusion method. A dose–response effect demonstrated that 5 or 10 μM DHA has no effect on Jurkat cell survival; however, 15 μM DHA rapidly decreased cell survival to 40% within 2 h of treatment. Cytotoxicity of 15 μM DHA was prevented by PA. Structurally similar phospholipids (lysophosphatidic acid, sphingosine 1-phosphate, sphingosine, and sphingosine phosphocholine) or metabolites of PA (lyso-PA and diacylglycerol) did not prevent DHA-induced cytotoxicity. PA did not produce micelles alone or in combination with DHA as examined spectrophotometrically, indicating that PA did not entrap DHA and therefore did not affect the amount of DHA available to the cells. Supporting this observation, the uptake or incorporation of [1-14C]DHA in Jurkat cells was not affected by the presence of PA. However, PA treatment reduced the amount of DHA-induced inorganic phosphate released from Jurkat leukemic cells and also inhibited DHA-induced dephosphorylation of cellular proteins. These observations indicate that PA has exerted its anti-cytotoxic effects by causing inhibition of protein phosphatase activities. Cytotoxicity of DHA on Jurkat cells was also blocked by the use of a highly specific caspase-3 inhibitor (N-acetyl-ala-ala-val-ala-leu-leu-pro-ala-val-leu-leu-ala-leu-leu-ala-pro-asp-glu-val-asp-CHO), indicating that the cytotoxic effects of DHA were due to the induction of apoptosis though activation of caspase-3. Consistent with these data, proteolytic activation of procaspase-3 was also evident when examined by immunoblotting. PA prevented procaspase-3 degradation in DHA-treated cells, indicating that PA causes inhibition of DHA-induced apoptosis in Jurkat leukemic cells. Since DHA-induced apoptosis can be inhibited by PA, we conclude that the process is mediated through activation of PP1.
Keywords: Docosahexaenoic acid; Phosphatidic acid; Protein phosphatase 1; Apoptosis; Jurkat cell; Cytotoxicity;

Second messengers regulate RGS2 expression which is targeted to the nucleus by Jaroslaw W. Zmijewski; Ling Song; Lualhati Harkins; Charles S. Cobbs; Richard S. Jope (201-211).
R egulators of G -protein S ignaling (RGS) proteins attenuate signaling activities of G proteins, and modulation of expression appears to be a primary mechanism for regulating RGS proteins. In human astrocytoma 1321N1 cells RGS2 expression was increased by activation of muscarinic receptors coupled to phosphoinositide signaling with carbachol, or by increased cyclic AMP production, demonstrating that both signaling systems can increase the expression of a RGS family member in a single cell type. Carbachol-stimulated increases in endogenous RGS2 protein levels appeared by immunocytochemical analysis to be largely confined to the nucleus, and this localization was confirmed by Western blot analysis which showed increased nuclear, but not cytosolic, RGS2 after carbachol treatment. Additionally, transiently expressed green fluorescent protein (GFP)-tagged, 6×His-tagged, or unmodified RGS2 resulted in a predominant nuclear localization, as well as a distinct accumulation of RGS2 along the plasma membrane. The intranuclear localization of GFP-RGS2 was confirmed with confocal microscopy. Thus, RGS2 expression is rapidly and transiently increased by phosphoinositide signaling and by cyclic AMP, and endogenous and transfected RGS2 is largely, although not entirely, localized in the nucleus.
Keywords: Regulator of G protein signaling; RGS2; G protein; Phosphoinositide signaling; Cyclic AMP;

Effect of p53 on centrosome amplification in prostate cancer cells by Xuesong Ouyang; Xianghong Wang; Kexin Xu; Dong-Yan Jin; A.L.M. Cheung; Sai Wah Tsao; Y.C. Wong (212-220).
Chromosomal instability (CIN) is one of the common features in prostate cancer, especially in advanced stages. Recently, the involvement of p53 in CIN through the regulation of centrosome amplification has been proposed in certain tumor types. In this study, we investigated the relationship between p53 and centrosome amplification in prostate cancer cells. Increased centrosome number and size were observed in DU145 and PC3 containing nonfunctional p53 compared to LNCap which expressed wild-type p53. Transfection of p53 into PC3 cells resulted in a decreased cell growth rate, G2/M arrest and decreased centrosome abnormalities. We provide the first evidence on a correlation between loss of p53 function and centrosome amplification in prostate cancer cells. Our results indicate that p53 may play a role in the regulation of centrosome amplification and loss of p53 may be one of the mechanisms involving CIN in prostate cancer cells.
Keywords: Prostate cancer; p53; Centrosome; G2/M cell cycle;

Cytokine production by macrophages in association with phagocytosis of etoposide-treated P388 cells in vitro and in vivo by Chizuru Kawagishi; Kahori Kurosaka; Naoko Watanabe; Yoshiro Kobayashi (221-230).
Chemotherapy and radiotherapy are performed for cancer patients with the hope that dying cancer cells are safely scavenged by phagocytic cells such as macrophages. In this study, we examined cytokine production by macrophages during and after the phagocytosis of etoposide-treated P388 cells in vitro and in vivo. Etoposide caused apoptosis as early as 5 h after treatment, as assessed as to the exposure of phosphatidylserine, increase in membrane permeability and DNA ladder formation. Phagocytosis by phorbol myristate acetate (PMA)-treated THP-1 cells occurred marginally when P388 cells were treated with etoposide for 10 h, while it occurred significantly with P388 cells treated for 24 h, as evidenced by flow cytometry and confocal microscopy. PMA-treated THP-1 cells produced pro-inflammatory cytokines, such as interleukin (IL)-1α, IL-8 and macrophage migration inhibitory factor (MIF), but not anti-inflammatory cytokines among those tested at the mRNA level during and after the phagocytosis of apoptotic cells. IL-8 and MIF were also produced at the protein level, and the IL-8 production was dependent on cell-to-cell contact when the plasma membranes of apoptotic cells were intact enough not to leak one of the cytoplasmic enzymes, lactate dehydrogenase. In addition, etoposide-treated P388 cells induced neutrophil infiltration as well as MIP-2 production upon injection into the peritoneal cavity of either normal mice or mice with sterile peritonitis. When macrophages ingesting and/or binding apoptotic P388 cells were isolated from the mice with sterile peritonitis using a cell sorter, they were found to produce MIP-2 upon culture.
Keywords: Apoptosis; Macrophage; Etoposide; Phagocytosis; Cytokine;

Caspase 7 can cleave tumor necrosis factor receptor-I (p60) at a non-consensus motif, in vitro by Douglas W. Ethell; Ella Bossy-Wetzel; Dale E. Bredesen (231-238).
Ligand binding to tumor necrosis factor receptor-I (TNFRI) can promote cell survival or activate the apoptotic caspase cascade. Cytoplasmic interaction of TNFRI with TRAF2 and RIP allows for the activation of JNK and NFκB pathways. Alternatively, a carboxy terminal death domain protein interaction motif can recruit TRADD, which then recruits FADD/MORT1, and finally procaspase 8. Aggregation of these components form a death inducing signaling complex, leading to the cleavage and activation of caspase 8. We have found that during apoptosis human TNFRI protein is lost in a caspase-dependent manner. The cytoplasmic tail of human TNFRI was found to be susceptible to caspase cleavage but not by caspase 8. Instead, the downstream executioner caspase 7 was the only caspase capable of cleaving TNFRI, in vitro. Identification and characterization of the cleavage site revealed a derivative of the classic EXD motif that incorporates a glutamate (E) in the P1 position. Using several criteria to establish that caspase activity was responsible for cleavage at this site, we confirmed that caspase 7 can cleave at a GELE motif. Mutation of the cleavage site prevented the apoptosis-associated cleavage of TNFRI. This ability of caspase 7 to cleave at a non-EXD or -DXXD motif suggests that the specificity of caspases may be broader than is currently held.
Keywords: Apoptosis; Caspase; Cell death; p60; Tumor necrosis factor receptor;

Author Index (239-240).