Establishment and characterization of a cell-line originated from human mucinous cystadenocarcinoma of the ovary

We recently established a cell line (designated 371M) derived from an ovarian mucinous cystadenocarcinoma. The tumor cells were obtained from the ascitic fluid of a 54-year-old Japanese woman while she was undergoing surgery. Adjuvant chemotherapy (combined paclitaxel and carboplatin) was administered, but was ineffective, and she died about 4 months after surgery. The 371M cells continuously propagated in vitro over a period of about 50 months and, to date, have undergone over 100 passages. They proliferated in a monolayered sheet with doubling times of 84 h and 37 h in the 10th and 34th passages, respectively. When transplanted into nude mice, the tumor histopathologically resembled the structure of the original tumor. The 371M cells secreted high levels of CA125 and CA19-9 into the culture medium. There were several abnormal chromosomes in all karyotypes selected at random. Sensitivity of 371M cells to a variety of anti-cancer drugs was examined by in vitro MTT assay, and the results suggested that CPT-11 and CDDP were more effective against 371M cells than other anti-cancer agents.     

Development of reconstituted embryos derived from transgenic embryonic stem cell nuclei

This study was carried out to transform embryonic stem (ES) cells and to produce the reconstituted embryos derived from transgenic ES cell nuclei. Then, in vitro/vivo developmental potency of transgenic ES cells were compared to that of control ES cells (non-transgenic ES cells) in the reconstituted embryos. Unfertilized B6D2F1 ooplasm at metaphase II (M II) and two kinds of ES cell lines, 129SV and transgenic (tg) 129SV transformed by EGFP gene, were used as nuclear recipients and nuclear donors, respectively. The M II chromosome-spindle complex was aspirated into the pipette with a minimal volume of ooplasm. After enucleation, the ES cell nuclei was injected into the enucleated ooplasm directly. Then, reconstituted embryos were activated in SrCl2, and they were cultured in HTF medium. There was no difference of developmental rate between reconstituted embryos derived from the control (non-transgenic) and the tg ES cells. From this result, we indicated that transgenic ES cells might not change the property of peculiarity of the ES cell by gene transfer. The expression of GFP gene in the embryos was observed by fluorescence microscope at the 4-cell and more stage. As comparison with development of the embryos derived from the control and tg ES cells, the difference of the development could not be confirmed between the two cell groups. When the reconstituted embryos derived from the control and tg ES cells were transferred into oviduct or uterus of pseudopregnant females, fetuses were observed 13.5 days post coitum. However, in all fetuses, developmental arrest and regression were seen 19.5 days post coitum.     

CO2 provides an intermediate link in the red light response of guard cells

Guard cells in intact leafs display light-induced membrane potential changes, which alter the direction of K+-transport across the plasma membrane (Roelfsema et al., 2001). A beam of blue light, but not red light, directed at the impaled guard cell triggers this response, while both light qualities induce opening of stomata. To gain insight into this apparent contradiction, we explored the possible interaction between red light and CO2. Guard cells in the intact plant were impaled with double-barrelled electrodes and illuminated with red light. Cells that were hyperpolarized in CO2-free air, depolarized after a switch to air with 700 micro l l(-1) CO2, in a reversible manner. As a result, K+-fluxes across the plasma membrane changed direction, to favour K+ extrusion and stomatal closure in the presence of CO2. Concurrent with the depolarization, an inward current across the plasma membrane appeared, most likely due to activation of anion channels. Guard cell responses to CO2 could be recorded in darkness as well as in red light. However, in darkness some cells spontaneously depolarized, these cells hyperpolarized again in red light. Here, red light was projected on a large area of the leaf and decreased the intracellular CO2 concentration by about 250 micro l l(-1), as measured with a miniature CO2 sensor placed in the substomatal cavity. We conclude, that in intact leaves the red light response of guard cells is mediated through a decrease of the intercellular CO2 concentration.     

Raman microspectroscopic study of biomolecular structure inside living adhesive cells

Cells adhesion is very important for many physiological processes. Using advanced Raman microspectroscopic technique, we selected T Leukemia cells (Jurkat) as the materials and obtained simultaneously conformation information of various biomolecules inside the whole living cells. By comparing the Raman microspectroscopic spectra of single and adhesive cancer cells, we found for the first time that when cells adhered, the conformation of the biomolecules (DNA, protein, carbohydrates and lipids) inside the cells had different changes: (i) the backbone of double-stranded DNA maintained orderly B-form or modified B-form conformation, whereas the groups of its deoxyribose and bases were modified; (ii) the conformational changes of the main chain and the side chain in the protein were obviously variant. The lines intensity belonging to a-helix and p-sheet decreased, while that of p-turn increased. Tyrosine and tryptophane residues of the protein changed from "buried state" to "exposed state"; the lines intensi     

Genetic retargeting of adenovirus vectors: functionality of targeting ligands and their influence on virus viability

Background

We studied the ability of adenovirus type 5 (Ad5) to encapsidate new cellular ligands carried by their fibers to yield functional retargeted vectors for gene therapy. Recombinant Ad5 fibers containing shaft repeats 1 to 7 and an extrinsic trimerization motif, and terminated by its native knob or amino acid motifs containing RGD, have been rescued into infectious virions.

Methods

Polypeptide ligands of cell surface molecules, including single‐chain antibodies or epidermal growth factor, were cloned into recombinant fibers. Phenotypic analysis of fiber constructs and rescuing into the Ad5 genome were performed. Recombinant viruses were characterized with reference to fiber content, growth rate and infectivity.

Results

A major limiting factor for recovering viable recombinant Ad5 carrying fiber‐fused polypeptide ligands was apparently the ability of the ligand to fold correctly within the cellular cytoplasm. This constraint has previously not been systematically evaluated in the literature. Phenotypic analysis of the fiber‐ligand fusions showed that their degree of cytoplasmic solubility correlated with their ability to yield viable Ad5 vectors. Our results suggested that the fiber manipulations diminish virus growth rate, probably through different, opposing effects: (i) the reduced shaft length increases fiber solubility in the absence of the knob but (ii) diminishes virus entry, and (iii) the absence of the knob alters the overall protein composition of the virion and decreases its fiber copy number.

Conclusions

Based on our findings, cytoplasmic solubility and cytoplasmic ligand reactivity of fiber‐ligand fusion proteins are the best prediction criterion for viability and recovery of genetically retargeted Ad vectors. Copyright © 2002 John Wiley & Sons, Ltd.

     

Modulation of apoptosis by HIV protease inhibitors

Advances in treatment have transformed the Human Immunodeficiency Virus (HIV) infection from a progressive and ultimately fatal disease to one that can be managed effectively by chronic suppressive antiretroviral therapy. The drugs now used to treat HIV infection not only inhibit viral replication but also have effects on cellular metabolism and homeostasis. Of particular interest to cellular immunologists, members of the HIV Protease Inhibitor (PI) class of antiretroviral agents possess intrinsic immunomodulatory and antiapoptotic properties. This review focuses on the development and use of PI together with their impact on HIV disease, immunity, and apoptosis.     

Mitochondrial permeability transition as a novel principle of hepatorenal toxicity in vivo

Atractyloside (Atr) binds to the adenine nucleotide translocator (ANT) and inhibits ANT-mediated ATP/ADP exchange on the inner mitochondrial membrane. In addition, Atr can trigger opening of a non-specific ion channel, within the ANT-containing permeability transition pore complex (PTPC), which is subject to redox regulation and inhibited by cyclosporin A (CsA). Here we show that the cytotoxic effects of Atr, both in vivo and in vitro, are determined by its capacity to induce PTPC opening and consequent mitochondrial membrane permeabilization (MMP). Thus, the Atr-induced MMP and death of cultured liver cells are both inhibited by CsA as well as by glutathione (GSH) and enhanced by GSH depletion. Similarly, the hepatorenal toxicity of Atr, assessed in vivo, was reduced by treating mice with CsA or a diet rich in sulfur amino acids, a regime which enhances mitochondrial GSH levels. Atr injection induced MMP in hepatocytes and proximal renal tubular cells, and MMP was reduced by either CsA or GSH. Acetaminophen (paracetamol)-induced acute poisoning was also attenuated by CsA and GSH, both in vitro and in vivo. Altogether these data indicate that PTPC-mediated MMP may determine the hepatorenal toxicity of xenobiotics in vivo.     

Overexpression of the type II transforming growth factor-beta receptor inhibits fibroblasts proliferation and activates extracellular signal regulated kinase and c-Jun N-terminal kinase

Transforming growth factor-beta (TGF-beta) is a bimodal regulator of cellular growth. The cellular effects of TGF-beta depend on the intensity of signals emanating from TGF-beta receptors. Low levels of receptor activity are sufficient to stimulate cell proliferation, while higher degrees of receptor activation are associated with growth inhibition. To study the mechanisms of these effects, a tetracycline-inducible expression system was used to overexpress type II TGF-beta receptors in NIH 3T3 fibroblasts. Overexpressed type II TGF-beta receptors suppressed fibroblast proliferation elicited by TGF-beta1, fibroblast growth factor (FGF) or platelet-derived growth factor (PDGF). Accompanying these anti-proliferative effects, increases in extracellular-signal regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) activity were detected. Furthermore, PDGF alpha-, but not PDGF beta-receptor protein levels were reduced by type II TGF-beta receptor overexpression. In conclusion, our system is an excellent tool to study the molecular mechanisms of growth inhibition by TGF-beta in fibroblasts. Activation of JNK and ERK, or modulation of PDGF receptor expression may be involved in this process.     

Paracrine action of transforming growth factor-alpha in rectal crypt epithelium of humans

Colon and rectal mucosal crypt epithelium is a rapidly renewing cell population, where cell proliferation is normally balanced by cell loss. This report concerns the putative paracrine action of transforming growth factor alpha(TGF-alpha) in this homeostatic process. Immunohistochemical staining for proliferating cell nuclear antigen (PCNA) and TGF-alpha was performed on biopsy specimens of rectal mucosa taken from consenting patients. The height of the proliferative compartment in mid-axially sectioned crypts in each individual was determined from the distribution of PCNA stained cells. The number of TGF-alpha stained cells that exhibited intense positive staining in a continuous column from the mouth down the side of the crypt was also scored in each individual patient. There was a significant positive correlation (P=0.05,n =22 patients) between the height of the proliferative compartment and the number of cells staining for TGF-alpha. Non-cellular TGF-alpha reactivity was also observed in the lamina propria adjacent to the TGF-alpha reactive epithelial cells, indicating secretion of TGF-alpha by these epithelial cells. These findings suggest that TGF-alpha is released from epithelial cells in the upper compartment of the crypt into the adjacent lamina propria and then diffuses to the epithelial cells in the lower part of the crypt, resulting in expansion of the proliferative compartment.     

The significance of alpha 5 beta 1 integrin dependent and independent actin cytoskelton organization in cell transformation and survival

Cell-matrix and cell-cell interactions are important physiological determinants of cell growth, survival and transformation. Cell adhesion to the extra cellular matrix (ECM) via integrins also crucially influences the organization of the cytoskeleton. It triggers a cascade of intracellular biochemical events, which regulate cell viability and growth. We have studied the relationship between cell attachment to the substratum and cytoskeletal organization and cell survival and transformation. Our results demonstrate that in the absence of attachment to the substratum, adhesion-dependent fibroblasts exhibit rapid loss of viability. However, a small percentage of cells survive even after remaining non-adherent for 16h. The adherent and non-adherent cells differ from one another both morphologically and physiologically. The latter show a loss of alpha5beta1 integrin expression on their surface and bind non-specifically to the substratum and ECM, thereby activating certain pathways more efficiently than adherent cells. We have also shown that non-adherent cells grow faster and have worse cytoskeletal organization after attachment to the substratum, and do not form focal adhesions or actin stress fibres. Hence, our data suggests that rat fibroblasts in prolonged suspension exhibit some properties that are comparable to cells undergoing transformation, by adapting integrin-dependent or independent signalling pathways for their survival.