Thermal enhancement of oxaliplatin-induced inhibition of cell proliferation and cell cycle progression in human carcinoma cell lines.

Hyperthermia is used to treat intraperitoneal colorectal carcinomatosis. In this setting, the molecular effects of oxaliplatin and hyperthermia, in combination and alone, were deciphered in ovarian and colon cancer cells. The combined antiproliferative effects of hyperthermia and oxaliplatin (Eloxatine) on human IGROV-1 ovarian carcinoma, Caco-2 and HT-29 colon carcinoma cell lines were investigated by cell viability test, cell cycle analysis and modulation of expression of cell cycle-related proteins. Oxaliplatin inhibited growth of all cell lines in a dose-dependent manner. The efficacy of the drug was markedly enhanced by concurrent exposure to mild heat shock (1 h, 42 degree C). In IGROV-1 cells, a low concentration (15 microg/ml) of oxaliplatin in combination with hyperthermia induced a transient G2/M arrest. In both colon carcinoma cell lines, a G1/S arrest with a reduction of the G0/G1 population occurred. In IGROV-1 and Caco-2 cells, growth arrest was accompanied by apoptosis as suggested by the appearance of sub-G1 population. Time-course changes of cell cycle regulatory proteins levels revealed accumulation of cyclins A and B as well as of cdc2 and cdk2 upon exposure of IGROV-1 cells to hyperthermia and oxaliplatin. In this cell line, p53 appeared to be implicated in both G2/M arrest and apoptosis. G1/S arrest of HT-29 cells was linked to up-regulation of cyclin E and p27(Kip1) and accumulation of the hypophosphorylated form of pRB, whereas in Caco-2 cells only the hyperphosphorylated form was detected as well as a down-regulation of the proto-oncogene c-myc. Taken together, the results of these in vitro studies suggest that hyperthermia and oxaliplatin might elicit antiproliferative effects by modulating the expression of cell cycle regulatory proteins through different signalling pathways.

The nuclear pore complex is involved in nuclear transfer of plasmid DNA condensed with an oligolysine-RGD peptide containing nuclear localisation properties.

One of the major barriers to efficient gene transfer and expression of nonviral vectors for gene therapy is passage across the nuclear envelope. We have previously shown that an oligolysine-RGD peptide that condenses plasmid DNA and binds to cell surface integrins can mediate increased internalisation of plasmid DNA into cells and synergistic enhancement of gene expression when complexed to a cationic lipid. In this report, we show that this enhancement is due to increased nuclear transfer of the plasmid DNA. We have applied the digitonin-permeabilised cell system that has been well established for the study of the nuclear transport of proteins to examine the nuclear transfer of plasmid DNA. Nuclear transfer of plasmid DNA complexed to an oligolysine-RGD peptide and lipofectamine appears to be an energy-dependent process involving the nuclear pore complex, since it is inhibited at 4 degrees C and by treatment with wheat germ agglutinin or with an antibody to the nuclear pore complex which all block nuclear protein import. In accordance with active nuclear transport, we have shown that all these treatments inhibit expression of a luciferase reporter plasmid in permeabilised cells. Nuclear transfer of pDNA is enhanced in mitotic cells, but cell division is not a prerequisite for transfer. We propose that the oligolysine-RGD peptide acts as a nuclear localisation signal and that the cationic lipid is more important for cell entry and endosome destabilisation than nuclear transfer.

Butyrate and trichostatin A effects on the proliferation/differentiation of human intestinal epithelial cells: induction of cyclin D3 and p21 expression.

BACKGROUND: Sodium butyrate, a product of colonic bacterial fermentation, is able to inhibit cell proliferation and to stimulate cell differentiation of colonic epithelial cell lines. It has been proposed that these cellular effects could be linked to its ability to cause hyperacetylation of histone through the inhibition of histone deacetylase. AIM: To analyse the molecular mechanisms of butyrate action on cell proliferation/differentiation and to compare them with those of trichostatin A, a well known inhibitor of histone deacetylase. METHODS: HT-29 cells were grown in the absence or presence of butyrate or trichostatin A. Cell proliferation and cell cycle distribution were studied after DNA staining by crystal violet and propidium iodide respectively. Cell cycle regulatory proteins were studied by western blot and reverse transcription-polymerase chain reaction. Cell differentiation was followed by measuring brush border enzyme activities. Histone acetylation was studied by acid/urea/Triton acrylamide gel electrophoresis. RESULTS: Butyrate blocked cells mainly in the G(1) phase of the cell cycle, whereas trichostatin A was inhibitory in both G(1) and G(2) phases. Butyrate inhibited the mRNA expression of cyclin D1 without affecting its protein expression and stimulated the protein expression of cyclin D3 without affecting its mRNA expression. Trichostatin A showed similar effects on cyclin D1 and D3. Butyrate and trichostatin A stimulated p21 expression both at the mRNA and protein levels, whereas their effects on the expression of cyclin dependent kinases were slightly different. Moreover, butyrate strongly stimulated the activity of alkaline phosphatase and dipeptidyl peptidase IV, whereas trichostatin A had no effect. Finally, a six hour exposure to butyrate or trichostatin A induced histone H4 hyperacetylation. At 15 and 24 hours, histone H4 remained hyperacetylated in the presence of butyrate, whereas it returned to control levels in the presence of trichostatin A. CONCLUSIONS: The data may explain how butyrate acts on cell proliferation/differentiation, and they show that trichostatin A does not reproduce every effect of butyrate, mainly because of its shorter half life.

Cell delivery, intracellular trafficking and expression of an integrin-mediated gene transfer vector in tracheal epithelial cells.

The mechanism of cell entry and intracellular fate of a gene transfer vector composed of a receptor-targeting, DNA-condensing peptide, RGD-oligolysine, a luciferase encoding plasmid DNA (pDNA) and a cationic liposome was examined. We demonstrate by confocal microscopy, electron microscopy and subcellular fractionation that the major mechanism of entry of the vector is endocytic. The vector complex rapidly (5 min) internalizes into early endosomes, then late endosomes and lysosomes. Entry involves, at least in part, clathrin-coated pit-mediated endocytosis since different conditions or drugs known to influence this pathway modify both uptake of pDNA and its expression. The observed increase in expression with addition of a lip some correlated with an increase in the rate of transfer of the pDNA to lysosomes, a decrease in intracellular recycling and exocytosis of the pDNA and an increase in the amount of pDNA in the nuclear fraction. Trafficking within the cell involved endosome fusion and the acid environment of the endosomes-lysosomes was beneficial for expression. After 30 min both the peptide and pDNA localized to the nucleus and the amount of intact pDNA in the nuclear fraction was highest with liposome and peptide. A better understanding of the cellular mechanisms by which vectors transfer to and traffic in cells should help design improved vectors.

Liposomes enhance delivery and expression of an RGD-oligolysine gene transfer vector in human tracheal cells.

Nonviral gene delivery systems consist predominantly of lipoplexes or receptor-targeting and nontargeting polyplexes. We examined integrin-mediated gene delivery using an Arg-Gly-Asp/oligo-L-lysine ([K]16RGD) cyclic peptide and investigated its gene transfer efficiency when associated with a cationic liposome. We demonstrated that human cystic fibrosis and noncystic fibrosis tracheal epithelial cells in culture express integrins that recognise the RGD integrin-binding motif. We found a 10-fold (P < 0.01) increased expression of a luciferase encoding plasmid in these cells when complexing the plasmid to the [K]16RGD peptide as compared with plasmid alone. This increase was specific to the [K]16RGD peptide since neither a [K]16RGE nor a [K]16 peptide gave a comparable increase. Expression was further enhanced 30-fold (P < 0.01) with lipofectamine and the ratio of DNA/peptide/lipofectamine was critical for specificity and expression. Fluorescence and radioactive labelling of the complex showed that the [K]16RGD peptide increased the endocytic uptake of DNA into cells. The cell association of both DNA and peptide increased even further with lipofectamine. Confocal microscopy showed that the [K]16RGD peptide and the DNA internalised together within 30 min and localised to vesicles in the perinuclear region. These results show that an integrin-binding ligand can deliver genetic material to airway cells and that a cationic liposome can enhance the efficacy of this nonviral vector system.

Enterocytic differentiation of the human Caco-2 cell line correlates with alterations in integrin signaling.

We previously reported that the enterocytic differentiation of human colonic Caco-2 cells correlated with down-regulation of fibronectin (FN) and laminin (LN), two extracellular matrix components interacting with cell surface integrin receptors. We now investigated whether Caco-2 cell differentiation was associated with alterations in integrin signaling with special interest in the expression and activity of focal adhesion kinase (FAK) and mitogen-activated protein (MAP) kinase. The differentiation of Caco-2 cells was associated with: 1) down-regulation of beta1 integrin expression at the mRNA and protein levels; 2) increased FAK expression together with decreased FAK autophosphorylation; 3) decreased FAK's ability to associate with PI3-kinase and pp60c-src; and 4) increased MAP kinase expression along with decreased MAP activity. In addition, we show that FAK and MAP kinase belong to distinct integrin signaling pathways and that both pathways remain functional during Caco-2 cell differentiation since the coating of differentiating cells on FN and LN but not on polylysine increased the tyrosine phosphorylation of FAK and of its endogenous substrate paxillin, and stimulated MAP kinase activity. In conclusion, our results provide evidence that FAK and MAP kinase, two signaling molecules activated independently by beta1 integrins in Caco-2 cells, undergo alterations of both expression and activity during the enterocytic differentiation of this cell line.

Compensatory apoptosis in response to SV40 large T antigen expression in the liver.

Transgenesis allows the in vivo determination of the effects of oncogene expression in normal tissues. In an attempt to understand the mechanism underlying liver transformation, we have previously created transgenic mice carrying the SV40 early gene sequences, which developed hepatocarcinoma in a reproducible way. In the present study, we show that constant expression of the transgene was directly correlated to an abnormally increased hepatocyte proliferation, even at the adult stage. We further demonstrate in this model that the preneoplastic stage of hepatocarcinoma is characterized by marked ploidy alterations as early as 1 month, including the emergence of aneuploid and hyperpolyploid cells, and the persistence of an important diploid cell population. We show that this elevated proliferation is early and transiently counterbalanced by a mechanism of apoptosis, which maintains liver homeostasis. The disappearance of this programmed cell death response effective during preneoplasia might signal the commitment of the liver to neoplasia.

Desferrioxamine regulates tumor necrosis factor release in mesangial cells.

Cultured rat mesangial cells have been demonstrated to express tumor necrosis factor alpha (TNF alpha) mRNA and to release TNF activity into the medium upon stimulation by bacterial lipopolysaccharide (LPS). The present study was undertaken to determine whether TNF was only secreted by mesangial cells or was also present as a cell-associated molecule. LPS-activated mesangial cells which had been fixed in paraformaldehyde lysed the TNF-sensitive L-929 fibroblasts, as assessed by 51Cr release. This cytotoxic activity was inhibited by anti-TNF alpha antiserum. Cell-associated TNF expression was demonstrable after less than one hour of exposure to LPS, peaked at two hours and decreased progressively thereafter, while TNF activity increased in the medium. Mesangial cell-associated TNF was localized at the cell surface, as shown by immunohistochemical demonstration and by the ability of plasma membranes purified from LPS-activated mesangial cells to lyse L-929 fibroblasts. Flow cytometry experiments revealed that two-thirds of LPS-activated mesangial cells were stained by anti-TNF alpha antiserum. The major part of these cell-associated TNF molecules persisted after low pH treatment, indicating that they were integral membrane proteins. As assessed by immunoprecipitation analysis, these proteins were 26 kDa molecules, whereas the released forms of TNF were 17 kDa molecules. Pretreatment of mesangial cells with desferrioxamine (DFX), an iron chelator preventing the synthesis of hydroxyl radicals (OH.), delayed the release of TNF from the membranes into the medium, and enhanced its cell surface expression. It also subsequently accelerated its decay in the medium.(ABSTRACT TRUNCATED AT 250 WORDS)

A serologic study of red cells and sera from 18 Rh:32,-46 (RN/RN) persons.

The red cells (RBCs) and sera from 18 RN/RN persons were studied. The study confirmed the Rh type D+C+E-c-e+Cw-, which is characterized by an increased expression of the D antigen; a markedly decreased expression of the C and e antigens; the presence of a low-incidence antigen, Rh32; and the absence of a high-incidence antigen, Rh46, which is associated with an epitope recognized by a murine monoclonal antibody (MR432). Other Rh antigens of low and high incidence were investigated, and the presence of Rh17 and Rh44 on the RBCs was confirmed. Three persons exposed to Rh:46 cells by pregnancy or transfusion (or both) had anti-Rh46. This antibody gave positive reactions with all RBCs of common and rare Rh phenotype except Rhnull, and those of D--, D.., DCw-, and RN homozygotes. This antibody is considered to be of clinical significance in case of transfusion or pregnancy.

False paternity exclusion in the Rh system: use of cytofluorometry analysis.

In a two-men paternity testing analysis, the first putative father was definitely excluded by six blood group systems; the second one was apparently excluded in the Rh system. In fact, an analysis by flow-cytometry demonstrated that this false exclusion was due to the presence of a D--haplotype in the father and child.

Mitochondrial uptake of rhodamine 123 by rabbit articular chondrocytes.

Rhodamine 123 was used to stain and analyze by flow cytometry the mitochondria of rabbit articular chondrocytes. Stationary primary cultures and exponentially growing subcultures were compared to enzymatically released chondrocytes from cartilage. The increase in mitochondrial fluorescence, when chondrocytes are transferred from cartilage to culture environment, is suggestive of some change in chondrocyte adaptation and/or differentiation in these conditions.

Histiocytosis X. Purified (T6+) cells from bone granuloma produce interleukin 1 and prostaglandin E2 in culture.

We have investigated the secretory function of cell suspensions from bone eosinophilic granulomas surgically collected in two patients with histiocytosis X. Unseparated cell preparations spontaneously produced interleukin 1 (IL-1) and prostaglandin E2 (PGE2). In order to ascertain that this secretion was due to the characteristic Langerhans cell-like histiocytosis X cells predominantly found in the bone lesions, we have purified T6+ cells by the use of a fluorescence-activated cell sorter. Such highly purified cell preparations were found to secrete IL-1 and PGE2 spontaneously in culture. Stimulation with endotoxins and treatment with interferon gamma (IFN gamma) revealed an intense IL-1 secretory function of histiocytosis X cells. Since both IL-1 and PGE2 are able to induce bone resorption in vitro, our findings are compatible with the hypothesis that histiocytosis X cells are responsible for the typical osteolytic lesion observed in histiocytosis X through the local secretion of these two mediators.