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. It also revealed energy homo-transfer within aggregates that TK366GFP progressively formed. Using a symmetric dimer model, structural parameters were estimated; the mutual orientation of the transition dipoles of the two GFP chromophores, calculated from the residual anisotropy, was 44.6 +/- 1.6 degrees, and the upper intermolecular limit between the two fluorescent tags, calculated from the energy transfer rate, was 70 A. Acquisition of the fluorescence steady-state intensity, lifetime, and anisotropy decay in the same cells, at different times after transfection, indicated that TK366GFP was initially in a monomeric state and then formed dimers that grew into aggregates. Picosecond time-resolved fluorescence anisotropy microscopy opens a promising avenue for obtaining structural information on proteins in individual living cells, even when expression levels are very low.

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. During these stages, mitochondrion morphology was fully preserved. In contrast, no chromatin condensation was observed after a rapid and total dissipation of mt delta psi. These results suggest that a partial decrease of mt delta psi would allow mitochondrial functions required to trigger apoptosis to be sustained.

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. The combination of fluorescence kinetic and fluorescence anisotropy dynamics indicates that the torsional dynamics of nuclear DNA is highly restrained in living cells.

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. Ethidium fluorescence variations have been used to probe DNA structural changes. This showed that: i) electrochemical gradient perturbations induced mtDNA structural change; ii) this change was readily reversible following a total but short collapse of the electrochemical gradient; iii) in contrast, a short and weak perturbation of the electrochemical gradient stabilized the mtDNA structural change; and iv) the degree of weak depolarization varied from cell to cell, showing the necessity of studying the effect of energetic perturbations at the level of an individual cell.

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. To get further information on the interaction between HIV and B cells, the susceptibility of several EBV-positive or -negative B cell lines to infection by SI and NSI isolates was checked. Following infection of an EBV- CD4+ CXCR4+ CCR5- B cell line (DG75) by an SI HIV-1 isolate, we obtained a cell line which chronically produced low-level infectious HIV-1 for 2 years (HIV-DG75). Immunocytochemical data, combined with in situ PCR data, established that HIV-DG75 cells consist of at least three populations uninfected cells, infected virus-producing cells, and infected but nonproducing cells. Moreover, HIV-DG75 cells which express p24 antigen do not go into apoptosis, contrary to T lymphocytes. We infer from these results that B cells could constitute a reservoir of infectious virus in infected patients.

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. After removal of the inhibitors or ionophores, ethidium fluorescence rapidly reappeared in mitochondria, together with the membrane potential. The fluorescence extinction did not result from an equilibrium shift caused by leakage of free ethidium out of mitochondria when the membrane potential was abolished but was most likely due to a dynamical mtDNA change that exposed intercalated ethidium to quencher, either by weakening the ethidium binding constant or by giving access of a proton acceptor (such as water) to the interior of mtDNA. Double labeling with ethidium and with a minor groove probe (4',6-diamino-2-phenylindole) indicated that mtDNA maintains a double-stranded structure. The two double-stranded DNA states, revealed by the fluorescence of mitochondrial ethidium, enhanced or quenched in the presence of ethidium, seem to coexist in mitochondria of unperturbed fibroblast cells, suggesting a spontaneous dynamical change of mtDNA molecules. Therefore, the ethidium fluorescence variation allows changes of DNA to be followed, a property that has to be taken into consideration when using this intercalator for in vivo as well as in vitro imaging studies.

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. RESULTS: When grown with fibroblasts, all three tumor cell lines showed increased clonogenicity and increased radioresistance. The radioprotective effect was maximal at a density of approximately 10(5) fibroblasts/100 mm Petri dish, and was greatest in the intrinsically radiosensitive tumor cell line. On the other hand, the effects of incubation with conditioned medium or on a fibroblastic matrix varied among the tumor cell lines. Thus, the protective effect afforded by coculture with fibroblasts must involve several cellular factors related to the fibroblast itself. CONCLUSIONS: These observations emphasize the importance of cultural conditions on the apparent radiosensitivity of human tumor cell lines, and suggest that the fibroblastic connective tissue enveloping the malignant cells should be considered when the aim is to establish a radiopredictive assay from surgical tumor fragments.

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. We describe here an assay that is specific for RecA activity even in bacterial crude extracts. In this assay, the ssDNA is bound to a nitrocellulose membrane, proteins are loaded on this membrane and it is then incubated with a labeled homologous dsDNA. Joint molecules are visualized by autoradiography. We have shown that, despite the reduced mobility of the DNA due to its binding to the membrane, RecA protein is able to promote formation of stable plectonemic joints, in a homology dependent manner. Fourteen other proteins involved in DNA metabolism were checked and did not produce a signal in our assay. Moreover, in Dot blot analysis as well as after native electrophoresis and electrotransfer on a ssDNA coated membrane, production of a signal was strictly dependent on the presence of active RecA protein in the bacterial crude extracts used. We named this assay Pairing On Membrane blot (POM blot).

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. We first confirm that a short heterologous insert (8bp) close to the break on the recipient cleaved M13 DNA inhibits recombination with circular as well as with linear donor molecules. The results indicate that, with these substrates, recombination is initiated at the level of the break, requires uninterrupted homology on both sides of the break, and is associated with a decreasing gradient of gene conversion. When the heterologous insertion is located on the plasmid donor DNA, similar results are obtained with a circular donor DNA. In contrast, with a linear donor molecule, bearing the insert, homology requirements, in the region of the break in M13 DNA, are abolished. This last result suggests that recombination could be initiated at the extremities of the linear donor DNA.

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. Recombination with the plasmid enhances the number of plaques and also the frequency of M13 producing blue plaques. Heterologous insertions in the region surrounding the break were analyzed for their effects on initiation of recombination. The extent of repair by recombination (number of plaques) was compared with the number of blue plaques among the repaired population. Initiation of recombination is inhibited when heterologous insertions are located at 7bp from the break, on the right side as well as on the left side. A low level of recombination is measurable for 27 bp of homology but the maximum efficiency of recombination occurred with homologies of 165 or 320 bp from the break to the heterologous insertion. At 320 bp, the extent of recombinational repair remained at a plateau level but the frequency of blue plaques progressively decreases. We have also analyzed the effect of different sizes of inserts. With longer inserts, a longer length of homology adjacent to the break is required for optimum recombination. However, the size of the insert does not affect the low level of recombination that occurred with a short homology (27 bp). The results indicate that the process is initiated at or near the break, requires homology on both sides of the break and is followed by an elongation from the double strand break to the distal regions of the DNA. Our data provide some support to the double-strand-break repair model established for meiotic recombination in yeast.

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. This nonpermissive state of the chromatin appears to be a characteristic feature of the nucleus in living cells. AO intercalation into DNA can be mediated by raising the nuclear Na+ concentration. This was achieved here by using a cation carrier, monensin, a procedure which permits the avoidance of cell permeabilization. Furthermore, we show that the discharge of lysosomal enzymes in the living cell, via a targeted photodynamic reaction which occurs at high levels of light intensity, can also release the constraints which impede dye intercalation into nuclear DNA. In conclusion, studies carried out under conditions where intercalative dyes such as AO are able to stain the nuclear DNA have to be interpreted with caution and do not provide direct evidence on the structural state of native chromatin. The molecular origin of the absence of AO intercalation in chromatin of the living cell is discussed with regard to the restrained uncoiling of the double helix which is required for dye intercalation.

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. Kinetic data show that a long exposure to Adriamycin triggers its efflux from both the nucleus and the cytoplasm while stimulating its metabolism. Therefore, a long exposure time to the drug appears to trigger a process of cellular detoxification by favouring its excretion from the cells via increased metabolism.

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. The results obtained have shown that some radioactive copper atoms remained firmly bound to the DNA molecule. Their amount was of the same order when using two different DNA isolation methods for the two cell lines studied. No significant differences were found when 64Cu was used as CuCl2 or as porphyric complex.

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. Different data show that the cellular lethal event is a consequence of 64Cu transmutation and not of the irradiation by the simultaneously emitted beta- and beta+ particles. Liquid holding results show that the lethal event is irreparable. The decays of 67Cu, another radioisotope of copper, lead to cell death with the same exponential survival curve and the same lethal efficiency as for 64Cu, in spite of their different decay schemes. The lethal efficiency of both copper isotopes is close to that of 125I utilized in the form of iododeoxyuridine under the same experimental conditions as 64Cu and 67Cu. The high lethal efficiency of radioactive copper transmutations raises questions about the role in DNA functioning of copper atoms known to be present in trace amounts in this macromolecule. The lethal consequence of radioactive copper transmutations suggests that the copper atoms bound to DNA are essential for cellular functioning.

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. In comparison to other psoralens, 4,5',8-trimethylpsoralen (4,5',8-TMP) is one of the most photoreactive psoralens and it forms a relatively high proportion of DNA interstrand cross-links. TMP-damaged HSV is efficiently reactivated by multiple infection in human fibroblasts. The extent of multiplicity reactivation is greater in cells from FA donors (five strains tested) than in normal cells (three strains). Mutagenesis studied in the viral thymidine kinase locus revealed that: (i) spontaneous viral mutation rate is lower in FA than in normal cells; and (ii) under conditions of multiple infection, the mutation rate is either greater (normal cells) or unchanged (FA cells) in the progeny from psoralen-damaged HSV compared to that from untreated virus. Taken together, these observations suggest that the pathway underlying multiplicity reactivation of psoralen-damaged HSV is error-free in FA cells relative to normal 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. This protection was found to be strickly confined to the homologous sequences potentially implicated in recombination.

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. Zn++, however, had a small but consistent protective effect on the induction of all three endpoints by x-irradiation.