Early apoptosis-related changes triggered by HSV-1 in individual neuronlike cells.

Early events of apoptosis following HSV-1 infection were investigated at the single-cell level using intensified fluorescence digital-imaging microscopy. The results provide evidence that infection of differentiated ND7 neuronlike cells by HSV-1 triggers detectable alterations indicative of physiological changes associated with the early stages of apoptosis. Less than 1 h after infection with HSV-1 (KOS strain) or K26GFP (GFP being fused to HSV-1 capsid protein VP26) we observed (i) moderate decrease in mitochondrial membrane potential (about 20%), (ii) exposure of phosphatidyl serine, (iii) morphological change in the mitochondria that became spherical instead of filamentous, and (iv) activation of caspase-8.

Picosecond-hetero-FRET microscopy to probe protein-protein interactions in live cells.

By using a novel time- and space-correlated single-photon counting detector, we show that fluorescence resonance energy transfer (FRET) between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) fused to herpes simplex virus thymidine kinase (TK) monomers can be used to reveal homodimerization of TK in the nucleus and cytoplasm of live cells. However, the quantification of energy transfer was limited by the intrinsic biexponential fluorescence decay of the donor CFP (lifetimes of 1.3 +/- 0.

Incorporation of green fluorescent protein into the essential envelope glycoprotein B of herpes simplex virus type 1.

Herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) is a major virion component, essential for various steps of virus replication in cells, such as entry and maturation, and cell fusion. In addition, gB is a strong inducer of the immune response in humans and has been involved in neuropathogenesis. To analyze gB during infection, a recombinant HSV-1 was generated containing gB fused to the green fluorescent protein (GFP).

Homo-FRET microscopy in living cells to measure monomer-dimer transition of GFP-tagged proteins.

Fluorescence anisotropy decay microscopy was used to determine, in individual living cells, the spatial monomer-dimer distribution of proteins, as exemplified by herpes simplex virus thymidine kinase (TK) fused to green fluorescent protein (GFP). Accordingly, the fluorescence anisotropy dynamics of two fusion proteins (TK27GFP and TK366GFP) was recorded in the confocal mode by ultra-sensitive time-correlated single-photon counting. This provided a measurement of the rotational time of these proteins, which, by comparing with GFP, allowed the determination of their oligomeric state in both the cytoplasm and the nucleus.

A moderate but not total decrease of mitochondrial membrane potential triggers apoptosis in neuron-like cells.

The effects of various degrees of perturbation of the mitochondrial membrane potential (mt delta psi) on apoptosis was investigated by intensified fluorescence digital-imaging microscopy on neuron-like cells, ND7. Mt delta psi was either decreased by 40% by the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP 100 nM, 15 min) or completely collapsed (FCCP 10 microM, 60 min). A moderate decrease of mt delta psi induced a reduction of mitochondrial NADH, followed by exposure of phosphatidyl serine and then by chromatin condensation, 36% of nuclei being condensed 60 min after FCCP treatment.

Restrained torsional dynamics of nuclear DNA in living proliferative mammalian cells.

Physical parameters, describing the state of chromatinized DNA in living mammalian cells, were revealed by in situ fluorescence dynamic properties of ethidium in its free and intercalated states. The lifetimes and anisotropy decays of this cationic chromophore were measured within the nuclear domain, by using the ultra-sensitive time-correlated single-photon counting technique, confocal microscopy, and ultra-low probe concentrations. We found that, in living cells: 1) free ethidium molecules equilibrate between extracellular milieu and nucleus, demonstrating that the cation is naturally transported into the nucleus; 2) the intercalation of ethidium into chromatinized DNA is strongly inhibited, with relaxation of the inhibition after mild (digitonin) cell treatment; 3) intercalation sites are likely to be located in chromatin DNA; and 4) the fluorescence anisotropy relaxation of intercalated molecules is very slow.

A transient decrease of electrochemical gradient stabilizes DNA structural change in single mitochondria of living cells.

The effect of controlled and reversible perturbation of the electrochemical gradient on the structural changes of mitochondrial DNA has been studied in living cells by fluorescence microscopy. Electrochemical gradient perturbations were induced by the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone and quantified by measuring the mitochondrial membrane potential using tetramethyl rhodamine methyl ester. Under our experimental conditions, we have shown that ethidium fluorescence was mainly due to ethidium molecules intercalated in mtDNA.

Production of HIV-1 by human B cells infected in vitro: characterization of an EBV genome-negative B cell line chronically synthetizing a low level of HIV-1 after infection.

Human immunodeficiency virus type 1 (HIV-1) has tropism for helper T lymphocytes and cells of the monocyte/ macrophage lineages. HIV-1 can also infect other cell types, including B cells. We show here that 10% of fresh circulating B cells from HIV-1-seronegative donors (i) express the CD4 receptor and CCR5 and CXCR4, two recently described coreceptors for HIV-1 and (ii) are permissive to HIV-1 with de novo proviral DNA integration following ex vivo infection by either SI (syncytium-inducing) or NSI (non-syncytium-inducing) isolates.

Dynamical change of mitochondrial DNA induced in the living cell by perturbing the electrochemical gradient.

Digital-imaging microscopy was used in conditions that allowed the native state to be preserved and hence fluorescence variations of specific probes to be followed in the real time of living mammalian cells. Ethidium bromide was shown to enter into living cells and to intercalate stably into mitochondrial DNA (mtDNA), giving rise to high fluorescence. When the membrane potential or the pH gradient across the inner membrane was abolished by specific inhibitors or ionophores, the ethidium fluorescence disappeared from all mtDNA molecules within 2 min.

Modulation of clonogenicity, growth, and radiosensitivity of three human epidermoid tumor cell lines by a fibroblastic environment.

Purpose: To develop a model vitro system to examine the influence of fibroblasts on the growth and survival of human tumor cells after exposure to ionizing radiation.

Methods And Materials: The cell system of three epidermoid carcinoma cell lines derived from head and neck tumors having differing growth potentials and intrinsic radiosensitivities, as well as a low passage skin fibroblast strain from a normal human donor. The tumor cells were seeded for five days prior to exposure to radiation: (a) in the presence of different numbers of fibroblasts, (b) in conditioned medium from stationary fibroblast cultures, and (c) on an extracted fibroblastic matrix.

Homologous pairing between single-stranded DNA immobilized on a nitrocellulose membrane and duplex DNA is specific for RecA activity in bacterial crude extract.

Reaction between a circular single stranded and a linear double stranded DNA molecule (ssDNA and dsDNA) provides an efficient system to study recombination mediated by RecA protein. However, classical assays using reaction in solution require highly purified enzymes. This limits biochemical studies of mutant RecA proteins from Escherichia coli or of RecA proteins from other organisms.

Structural effect of donor DNA on the initiation of recombination for double strand break repair in human nuclear extracts.

The effect of the structure of donor DNA molecules on the initiation of recombination for double strand break repair in human nuclear extracts, was investigated here. A unique double strand break was introduced into M13 duplex derivatives by digestion with restriction enzymes. After coincubation of the cleaved DNA in human nuclear extracts, with a plasmid containing M13 sequences spanning the break, double strand break repair was estimated by the plating efficiency in JM109 (RecA1) bacteria.

Cytotoxic effects and pharmacokinetic analysis of combined adriamycin and X-ray treatments in human organotypic cell cultures.

Organotypic cultures of human A549 cells were used as a tumor model to investigate sequence effects for combination treatments with adriamycin (ADR) and X-irradiation. Initial drug exposure led to the greatest cytotoxic effect especially when X-rays were delivered 24 h later and this subsequent irradiation did not significantly modify the intracellular ADR concentration. In contrast, post-irradiation drug exposure gave rise to a lower cytotoxic effect, and induced a marked reduction of intracellular and more specifically intranuclear ADR uptake and retention, especially when the drug was given 24 h later.

A single 24h contact time with adriamycin provokes the emergence of resistant cells expressing the Gp 170 protein.

We investigate here, in A549 cells, the influence of a single short (1h) or long (24h) adriamycin (ADR) contact time on the long-term cytotoxicity of the drug and on the emergence of resistant cells. In contrast to a 1 h ADR contact, a 24 h treatment provokes the emergence of resistant cells overexpressing the Gp170 protein and this overexpression is maintained for at least three months without any drug selection pressure.

Directional recombination is initiated at a double strand break in human nuclear extracts.

The involvement of a double strand break in the initiation of homologous recombination was examined in human nuclear extracts. M13 duplex derivatives, containing inserts in the LacZ' region (producing white plaques), were cleaved by restriction enzymes and coincubated in the extracts with a circular plasmid containing the LacZ' region without insert, and unable to produce plaques. Repair was estimated by the ability to produce plaques after transfection into JM109 (recA1) bacteria.

Impossibility of acridine orange intercalation in nuclear DNA of the living cell.

The ability of a "vital" dye, acridine orange (AO), to intercalate into the DNA of living cells was investigated by quantitative intensified fluorescence microscopy and digital imaging under various conditions of dye concentration, excitation light intensity, and ionic concentration. Our results demonstrate that the bulk of chromatin DNA is packed in a way that does not allow AO intercalation. At low dye concentrations and very low levels of light intensity, the only fluorescent structures observed inside the nucleus are the nucleoli.

Adriamycin uptake and metabolism in organotypic culture of A549 human adenocarcinoma cells according to the exposure time.

In organotypic cultures (nodules) of A 549 human lung adenocarcinoma cells, the long-term cytotoxicity of Adriamycin is strongly improved by shortening the exposure time to the drug. In order to gain insight into the mechanisms of Adriamycin toxicity in this system, we have examined the drug uptake, retention and metabolism by fluorescence microscopy and HPLC analysis. A 549 nodules efficiently metabolize Adriamycin, two major metabolites, adriamycinol and an aglycone derivative, as yet chemically unidentified, are formed and efficiently excreted.

Biochemical studies of human (A549) and simian (CV1) cells labeled with 64Cu, 67Cu, and 125IUdR.

CV1 and A549 cells were grown in the presence of 64Cu porphyric complex, 64CuCl2, or 67CuCl2. Radioactive copper determinations were performed on whole cells and on isolated cellular DNA. 125IUdR was used to calibrate the particular extraction and purification procedures we developed because of the half-lives of 64Cu and 67Cu.

Similar lethal effect in mammalian cells for two radioisotopes of copper with different decay schemes, 64Cu and 67Cu.

The decays of 64Cu incorporated in human malignant (A549) or monkey nonmalignant (CVI) cells lead to cell death. When plotted as a function of the radioactivity introduced in the growth medium (microCi/ml at t = 0), the residual colony-forming capability decreases exponentially. The slope of the corresponding curve is steeper for A549 than for CV1 cells.

Molecular analysis of homologous recombination catalysed by human nuclear extract: fidelity and DNase protection.

We present a molecular analysis of DNA's resulting from homologous recombination, between two duplex molecules, and catalysed by human nuclear extracts. Sequence analysis of 20 recombined clones (400 nucleotides per clone), in a genetically silent sequence surrounding the recombination initiation or termination site, shows no modification compared to the parental sequence. Transient protection of the DNA's against DNase treatment was brought about by the nuclear extract.

Multiplicity reactivation and mutagenesis of trimethylpsoralen-damaged herpes virus in normal and Fanconi's anaemia cells.

Fanconi's anaemia (FA) cells are hypersensitive to the lethal effect of DNA cross-linking compounds. Herpes simplex virus (HSV) has been used here as a probe to monitor in FA cells repair of psoralen damage of which cross-links are a part. The replication of HSV is impaired when its DNA contains covalently photobound psoralen molecules.

Studies of mutagenesis and neoplastic transformation by bivalent metal ions and ionizing radiation.

We examined the influence of nontoxic concentrations of each of two essential (Zn++ and Mn++) and one nonessential (Ni++) bivalent metal ions on spontaneous and radiation-induced neoplastic transformation and specific gene mutations in mammalian cells. All three metals induced low levels of transformation in mouse BALB/3T3 cells but exerted no mutagenic effect in CHO cells (hprt locus) over a broad range of concentrations. Continuous incubation for 8 or 15 days with each of the metal ions did not enhance the frequency of cell killing, transformation, or mutations induced by acute exposure to x-rays.

The cytostatic efficiency of gamma-rays on organotypic cultures of malignant cells is greater with irradiation at 23 degrees C compared to 37 degrees C.

The cytostatic efficiency of gamma-rays was determined following irradiation at 23 degrees C or 37 degrees C on A549 human lung carcinoma cells, irradiated either in monolayers or in three-dimensional organotypic cultures (nodules) with a dose-rate of 0.5 Gy/min. For the monolayers, the residual colony forming ability of single cells, and for the nodules, the growth rate, the proportion of regenerating or disaggregating nodules and the growth rate of regenerated nodules were measured.