The role of histone acetylation versus DNA damage in drug-induced senescence and apoptosis.

The present study was undertaken to determine the significance of histone acetylation versus DNA damage in drug-induced irreversible growth arrest (senescence) and apoptosis. Cellular treatment with the DNA-damaging drugs doxorubicin and cisplatin or with the histone deacetylase inhibitor trichostatin A, led to the finding that all the three drugs induced senescence at concentrations significantly lower than those required for apoptosis. However, only doxorubicin and cisplatin induced activation of H2AX, a marker for double-strand break formation. Interestingly, this occurred mainly at apoptosis and not senescence-inducing drug concentrations, suggesting that non-DNA-damage pathways may be implicated in induction of senescence by these drugs. In agreement with this, chromatin immunoprecipitation experiments indicated that doxorubicin was able to induce acetylation of histone H3 at the promoter of p21/WAF1 only at senescence-inducing concentrations. Collectively, these findings suggest that alteration of chromatin structure by cytotoxic drugs may represent a key mediator of senescence.

Factors secreted by human neuroblastoma mediated doxorubicin resistance by activating STAT3 and inhibiting apoptosis.

BACKGROUND: The transcription factor Stat3 has been reported to play a key role in protecting cells against apoptosis by up-regulating expression of the anti-apoptotic gene BCl-xL. This investigation analyzes the relationship between the development of resistance to doxorubicin-mediated apoptosis in neuroblastoma cells (SKN-SH) and activation of the Stat3 signaling pathway. MATERIALS AND METHODS: A drug-resistant cell line (SKN-SH/Dox6) was generated by continuous exposure to incremental concentrations of doxorubicin. Specific antibodies were utilized for Western blots and confocal microscopy to determine the nuclear localization of activated Stat3. RESULTS: Doxorubicin-mediated DNA fragmentation was inhibited and caspase-3 activity decreased in SKN-SH/Dox6 cells. Up-regulation of Stat3 phosphorylation and Bcl-xL expression, increased nuclear translocation of phospho-Stat3, and binding to DNA occurred only in resistant SKN-SH/Dox6 cells. The expression of Bcl-xL was inhibited by AG490, an inhibitor of the JAK/Stat3 signaling pathway, suggesting that the regulation of Bcl-xL and Stat3 involved a common mechanism. Activation of Stat3 in SKN-SH/Dox6 cells was contingent upon stimulation evoked by ligands secreted by the drug-resistant cells. Evidence to support this hypothesis was provided by experiments in which doxorubicin-sensitive SKN-SH cells were preincubated with conditioned media obtained from doxorubicin-resistant SKN-SH/Dox6 cells. This treatment increased Stat3 activation. It also rendered SKN-SH cells resistant to doxorubicin as demonstrated by a sharp decrease in doxorubicin-induced DNA degradation and cytotoxic potency. CONCLUSIONS: These findings suggest that the resistance of SKN-SH/Dox6 cells to doxorubicin may be mediated by anti-apoptotic factor(s) that are synthesized and secreted by tumor cells in response to cytotoxic agents.

Doxorubicin-induced apoptosis in caspase-8-deficient neuroblastoma cells is mediated through direct action on mitochondria.

UNLABELLED: The induction of p53 expression and stimulation of the Fas/caspase-8 pathway represent major mechanisms by which cytotoxic drugs induce apoptosis, but in neuroblastomas, the caspase-8 gene is often not expressed. PURPOSE: The aim of this study was to determine whether doxorubicin could induce apoptosis in caspase-8-deficient neuroblastoma cells and to define its mechanism of action. METHODS: The caspase-8-deficient human neuroblastoma cell line, SKN-SH, was incubated with doxorubicin and the apoptotic response, as well as expression of apoptotic molecules in the p53/ Fas/caspase-8 pathway, were determined. RESULTS: SKN-SH cells incubated with doxorubicin readily underwent apoptosis in a concentration-dependent manner. Western blot analyses with specific antibodies demonstrated that both p53 and Fas ligand were endogenously expressed in SKN-SH cells, but their expression was not stimulated by doxorubicin. Fas receptor was not detected in these cells and caspase-8 was totally absent. Electron microscopic analyses of SKN-SH cells treated with doxorubicin revealed pronounced alterations in mitochondrial structure. This treatment also induced the release of cytochrome c from mitochondria and activated the downstream apoptotic intermediate, caspase-3. CONCLUSION: These results indicate that the p53/Fas/caspase-8 system does not play a role in mediating the apoptotic action of doxorubicin in the human neuroblastoma cell line SKN-SH. Thus, mitochondria and downstream apoptotic signaling intermediates may be considered as key targets for doxorubicin-induced apoptosis in neuroblastoma tumors having deficiencies in the Fas/caspase-8 system.

Expression of bisecting GlcNAc in pediatric brain tumors and its association with tumor cell response to vinblastine.

Increased expression of the bisecting GlcNAc has been correlated with tumor progression in several experimental tumor models. Its expression and function in brain tumors are, however, not yet known. In this study, we investigated expression of the bisecting GlcNAc structure in a series of pediatric brain tumors and its relationship to tumor response to vinblastine. A plant lectin (E-PHA) that recognizes the bisecting GlcNAc structure was used for detection of this molecule in a total of 90 pediatric brain tumors and normal brain tissue specimens. Our results showed that, whereas E-PHA staining was undetectable in the normal brain tissue, pediatric brain tumor specimens exhibited different levels of reactivity. Lectin staining was particularly prominent in high-grade astrocytomas (73%) and ependymomas (72%). In astrocytomas, there was a positive correlation with the tumor grade, which suggests that the bisecting GlcNAc may be of particular interest as a tumor marker for diagnosis and/or prognosis. By using a human glioma cell culture model, we have found that treatment of these cells with E-PHA lectin enhances their sensitivity to vinblastine. E-PHA interacted directly with the drug transporter P-glycoprotein and inhibited its drug efflux function. In a drug-resistant glioma cell line transfected with the mdr1 gene, drug resistance was reversed by E-PHA. Our findings indicate that: (a) expression of the bisecting GlcNAc in pediatric brain tumors may have a potential relevance as a tumor marker; and (b) glioma response to chemotherapy may be modulated through the bisecting GlcNAc.

Sialidase gene transfection enhances epidermal growth factor receptor activity in an epidermoid carcinoma cell line, A431.

Glycosphingolipids expressed in cancer cells have been implicated in the modulation of tumor cell growth through their interaction with transmembrane signaling molecules such as growth factor receptors. For glycosphingolipids to interact with growth factor receptors, the presence of sialic acid seems to be essential. Stable transfection of a gene encoding a soluble Mr 42,000 sialidase into a human epidermoid carcinoma cell line (A431) provided an approach by which the level of terminal lipid-bound sialic acid on the cell surface could be altered. In the sialidase-positive clones, the level of ganglioside GM3 was diminished, and little change was observed in protein sialylation. Sialidase-transfected cells grew faster than control cells. Sialidase expression did not modify the binding of epidermal growth factor (EGF) to its receptor but enhanced EGF receptor (EGFR) tyrosine autophosphorylation as compared to that of parental cells or cells transfected with the vector (pcDNA3) alone. Moreover, the phosphorylation of the EGFR, as well as other protein substrates, was observed at low EGF concentrations, suggesting an increase in the receptor kinase sensitivity. These data provided evidence that changes in ganglioside expression in cancer cells by appropriate gene transfection can dramatically affect EGFR kinase activity. Hence, the modulation of ganglioside expression may represent an approach to alter tumor cell growth.

Very low density lipoproteins and interleukin 2 enhance the immunogenicity of 9-O-acetyl-GD3 ganglioside in BALB/c mice.

Gangliosides expressed by tumor cells constitute potential targets for immunotherapy. A major limitation of protocols aiming to immunize patients against tumor gangliosides is the weak immunogenicity of these molecules. We have previously shown that exogenous gangliosides are essentially bound to serum lipoproteins. In this study we have analyzed the influence of human serum lipoproteins on the immunogenicity of purified human ganglioside 9-O-acetyl-GD3 in BALB/c mice. Although expressed at very low levels in mice, this ganglioside was not immunogenic when administered in the form of micelles. However 9-O-acetyl-GD3 adsorbed onto Very Low Density Lipoproteins (VLDL) was strongly and reproducibly immunogenic, inducing both an IgM and an IgG response, with higher titers than those obtained with total serum. The IgM antibody response appeared after a single injection whereas the IgG response was observed after 3 weeks but was stronger and more durable. The antibody response to 9-O-acetyl-GD3 bound to other serum fractions was weak or absent. The addition of recombinant interleukin 2 (IL-2) enhanced weak antibody responses to 9-O-acetyl-GD3 thereby facilitating responses to ganglioside in micelles and in protein-free Very Low Density Particles. Using in vitro assays, we demonstrated that VLDL-bound ganglioside 14C-GM3 was more sensitive to the effect of neuraminidase than gangliosides bound to other lipoprotein fractions, suggesting greater accessibility of VLDL-bound gangliosides. These results indicate that VLDL-bound gangliosides are the most immunologically active fraction of serum gangliosides. VLDL or similar particles and recombinant IL-2 may be useful adjuvants for immunization with gangliosides.

alpha2,6-Sialyltransferase gene transfection into a human glioma cell line (U373 MG) results in decreased invasivity.

Glycosyltransferase gene transfection into cell lines has been an approach used successfully to elucidate the functional role of cell surface glycoconjugates. We have transfected the rat CMP-NeuAc:Galbeta1,4GlcNAc alpha2,6-sialyltransferase (EC 2.4.99.1) gene into a human, tumorigenic, glioma cell line, U373 MG. This transfection led to a marked inhibition of invasivity, alterations in adhesivity to fibronectin and collagen matrices, and inappropriately sialylated alpha3beta1 integrin. Adhesion-mediated protein tyrosine phosphorylation was reduced in the transfectants despite increased expression of focal adhesion kinase, p125fak. Furthermore, the transfectants showed a distinct cell morphology, an increased number of focal adhesion sites, and different sensitivity to cytochalasin D treatment than control U373 MG cells. These results suggest that inappropriate sialylation of cell surface glycoconjugates, such as integrins, can change focal adhesion as well as adhesion-mediated signal transduction and block glioma cell invasivity in vitro.

Gene transfection-mediated overexpression of beta1,4-N-acetylglucosamine bisecting oligosaccharides in glioma cell line U373 MG inhibits epidermal growth factor receptor function.

N-linked oligosaccharides appear to be important for the function of the epidermal growth factor (EGF) receptor. In a previous study (Rebbaa, A., Yamamoto, H., Moskal, J. R., and Bremer, E. G. (1996) J. Neurochem. 67, 2265-2272), we showed that binding of the erythroagglutinating phytohemagglutin lectin from Phaseolus vulgaris to the bisecting structures on the EGF receptor from U373 MG glioma cells blocked EGF binding and receptor autophosphorylation. In this study we examined the consequences of overexpression of the bisecting structure on the EGF receptor by gene transfection of U373 MG cells with the N-acetylglucosaminyltransferase III (GnT-III). This modification leads to a significant decrease in EGF binding and EGF receptor autophosphorylation. In addition, the cellular response to EGF was found to be altered. Proliferation of U373 MG cells in serum-free medium is inhibited by EGF. In contrast, proliferation of the GnT-III-transfected cells was stimulated by EGF. These data demonstrate that changes in EGF receptor glycosylation by GnT-III transfection reduces the number of the active receptors in U373 MG cells and that this change results in change in the cellular response to EGF.

Binding of erythroagglutinating phytohemagglutinin lectin from Phaseolus vulgaris to the epidermal growth factor receptor inhibits receptor function in the human glioma cell line, U373 MG.

Little is known about the role of the N-linked oligosaccharides in the function of the epidermal growth factor (EGF) receptor (EGF-R). In a human glioma cell line, U373 MG, EGF-Rs contain the bisecting N-linked oligosaccharide sequence recognized by erythroagglutinating phytohemagglutinin lectin from Phaseolus vulgaris (E-PHA). Incubation of E-PHA with cultured U373 MG cells results in inhibition of EGF binding to its receptor and consequently inhibition of EGF-induced autophosphorylation of the receptor. Consistent with the inhibitory effects on the EGF-R, phenotypic events that depend on EGF-R signaling, such as cell spreading and proliferation, were also found to be modified. The effect of this lectin seems to be specific because leukoagglutinating phytohemagglutinin lectin from P. vulgaris (L-PHA), an isolectin of E-PHA, had no effect on EGF-R activity or the biological functions of these cells even though L-PHA was able to bind to the EGF-R. These findings suggest the presence of an important bisecting N-linked oligosaccharide structure in close proximity to the EGF binding site on the receptor. Furthermore, these results suggest the possibility that E-PHA lectin binding may provide an additional approach to blocking EGF-dependent glioma cell growth.

Ganglioside GM3 inhibition of EGF receptor mediated signal transduction.

Ganglioside GM3 is a membrane component that has been described to modulate cell growth through inhibition of EGF receptor associated tyrosine kinase. In order to determine if the inhibition of cell growth by this ganglioside is specifically mediated through EGF receptor signaling, the effects of GM3 on key enzymes implicated in EGF signaling were determined and compared to another inhibitor of the EGF receptor kinase. Treatment of A1S cells in culture by GM3 or a tyrosine kinase inhibitor, leflunomide, led to the inhibition of MAP kinase and PI3 kinase activities. There was no detectable effect on phosphotyrosine phosphatases. In a cell free system, however, GM3 had no effect on the activity of these signaling intermediates. Leflunomide was able to directly inhibit MAP kinase activity. GM3 and leflunomide were also found to act differently on the expression of the early immediate genes. The expression of c-fos and c-jun was inhibited by both GM3 and leflunomide. The expression of c-myc, however, was only inhibited by leflunomide. These findings suggest that the action of GM3 on cell growth and signaling is specifically mediated by EGF receptor and that this ganglioside does not act directly on the intracellular intermediates of EGF receptor signaling. In addition, soluble small molecule tyrosine kinase inhibitors such as leflunomide can directly affect the activity of MAP kinases and possibly other signaling intermediates. The direct effects of leflunomide on signaling intermediates may explain the differential effects of leflunomide and GM3 on gene expression and cell growth.

Optimal conditions to radiolabel (3H or 14C) aminosugar-containing glycosphingolipids by de-N-acetylation and re-N-acetylation.

The optimal conditions were examined for selective re-N-acetylation with 14C or 3H.acetic anhydride of de-N-acetylated aminosugar-containing glycosphingolipids. Re-N-acetylation, which is nearly quantitative within 10 minutes in methanol, occurs selectively up to a maximal 100% yield when using a molar ratio of 5 mol of acetic anhydride per mole of aminosugar present in the glycosphingolipid. Above this molar ratio, it was observed some O-acetylation of carbohydrates which could be removed by mild alkali treatment. The method allows the choice of 14C- or 3H-labeling of glycosphingolipids with a final specific radioactivity which depends solely on the one of acetic anhydride. The binding of specific antibodies to glycosphingolipids, which was abolished upon de-N-acetylation, was again detectable after re-N-acetylation with radioactive acetic anhydride, suggesting that the native structures were recovered. This procedure of radiolabeling offers safety, rapidity and broad applicability to alkali-stable aminosugar-containing glycosphingolipids.

Deficiency of ganglioside biosynthesis in metastatic human melanoma cells: relevance of CMP-NeuAc:LacCer alpha 2-3 sialyltransferase (GM3 synthase).

The glycosphingolipid patterns were analyzed on two clones derived from a human melanoma cell line and selected for their respectively high and low metastatic ability in immunosuppressed newborn rats. Conversely to the weakly metastatic cells which exhibited a pattern similar to that of the parental cell line, highly metastatic human melanoma cells appeared to be deficient in ganglioside biosynthesis. An accumulation of lactosylceramide was found in the latter cells, with low amounts of GM3 as the only ganglioside detected and a fourfold decreased activity of GM3 synthase (EC 2.4.99.9). After subcutaneous injection of metastatic cells in newborn rats, the cells proliferating in the tumor induced at the injection site re-expressed the four common gangliosides of melanoma: GM3, GM2, GD3 and GD2, whereas the cells growing in the lungs as metastatic nodules were deficient in ganglioside synthesis and showed an accumulation of lactosylceramide. Taken together, our results suggest that the human melanoma cells which are able to escape from the primary tumor and invade the lungs have an impaired ganglioside biosynthesis with a deficient GM3 synthase.

Inhibition of immune cell proliferation and cytokine production by lipoprotein-bound gangliosides.

We have analyzed the immunomodulatory effect of human melanoma gangliosides bound to serum lipoprotein fractions on normal human immune-competent cells in vitro. Total melanoma gangliosides in micelles inhibited proliferation of peripheral blood mononuclear cells stimulated by various mitogens, modulated lymphocyte surface molecules CD2, CD3, CD4, CD5 and CD8 and inhibited the production of interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha (TNF alpha) and IL-6 by stimulated adherent cells. Most of these effects were abrogated in the presence of serum. Purified serum lipoprotein fractions were tested for their ability to allow or inhibit the immunomodulatory effects of gangliosides. Melanoma gangliosides bound to very-low-density lipoproteins (VLDL) were shown to be as potent modulators of the immune response in vitro as when they were presented to cells in the form of micelles. Gangliosides bound to low-density lipoproteins were less active and gangliosides bound to high-density lipoproteins or the lipoprotein-free fraction had no immunomodulatory effects. Given the fact that gangliosides are predominantly bound to lipoproteins in serum, we conclude that lipoproteins are important determinants of the immunomodulating potential of tumor gangliosides, and that the immunomodulatory effects of melanoma gangliosides observed in vitro may also occur in vivo.

Characterization of a monoclonal antibody that specifically binds to choline phospholipids and its use in immunocytochemistry.

A monoclonal IgM (MC22-33F), raised in response to mouse embryonic dental papilla cells, was selected for further analysis on the basis of the unusual resistance of its epitope to detergent extractions and protease treatments of cell cultures. Binding of MC22-33F to cultured cells was abolished after either pre-treatment of the cells with phospholypase C or pre-incubation of the hybridoma culture supernatant with multilamellar phosphatidylcholine-containing vesicles. MC22-33F reacted with phosphatidylcholine, with the phosphatidylcholine analogue dimethylphosphatidylethanolamine, and with sphingomyelin immobilized on polystyrene surfaces or in thin-layer chromatograms. Crossreaction with other phospholipids was not observed. The surface of cultured epithelial cells was labeled by MC22-33F at sites of bleb formation. Combining immunostaining by MC22-33F and histochemical staining of cultured cells revealed codistribution of phospholipid-containing inclusions with either lysosomes or neutral fat droplets, and inhibition of lipid degradation by kanamycin resulted in a parallel accumulation of these inclusions and of neutral fats in the cytoplasm. Immunolabeling by MC22-33F of frozen mouse tissues was maximal in fat-storing and steroid-producing cells. Extracellular phospholipids present in calcifying cartilage septa strongly reacted with MC22-33F. This monoclonal antibody offers an interesting alternative to histochemical lipid stains for investigating fatty metamorphosis and extracellular lipid deposition under physiological and pathological conditions.