Characterization of a ribonuclease III-like protein required for cleavage of the pre-rRNA in the 3'ETS in Arabidopsis.

Ribonuclease III (RNaseIII) is responsible for processing and maturation of RNA precursors into functional rRNA, mRNA and other small RNA. In contrast to bacterial and yeast cells, higher eukaryotes contain at least three classes of RNaseIII, including class IV or dicer-like proteins. Here, we describe the functional characterization of AtRTL2, an Arabidopsis thaliana RNaseIII-like protein that belongs to a small family of genes distinct from the dicer family. We demonstrate that AtRTL2 is required for 3'external transcribed spacer (ETS) cleavage of the pre-rRNA in vivo. AtRTL2 localizes in the nucleus and cytoplasm, a nuclear export signal (NES) in the N-terminal sequence probably controlling AtRTL2 cellular localization. The modeled 3D structure of the RNaseIII domain of AtRTL2 is similar to the bacterial RNaseIII domain, suggesting a comparable catalytic mechanism. However, unlike bacterial RNaseIII, the AtRTL2 protein forms a highly salt-resistant homodimer that is only disrupted on treatment with DTT. These data indicate that AtRTL2 may use a dimeric mechanism to cleave double-stranded RNA, but unlike bacterial or yeast RNase III proteins, AtRTL2 forms homodimers through formation of disulfide bonds, suggesting that redox conditions may operate to regulate the activity of RNaseIII.

A chloroplastic RNA-binding protein is a new member of the PPR family.

P67, a new protein binding to a specific RNA probe, was purified from radish seedlings [Echeverria, M. and Lahmy, S. (1995) Nucleic Acids Res. 23, 4963-4970]. Amino acid sequence information obtained from P67 microsequencing allowed the isolation of genes encoding P67 in radish and Airabidopsis thaliana. Immunolocalisation experiments in transfected protoplasts demonstrated that this protein is addressed to the chloroplast. The RNA-binding activity of recombinant P67 was found to be similar to that of the native protein. A significant similarity with the maize protein CRP1 [Fisk, D.G., Walker, M.B. and Barkan, A. (1999) EMBO J. 18, 2621-2630] suggests that P67 belongs to the PPR family and could be involved in chloroplast RNA processing.

Two ribosomal DNA-binding factors interact with a cluster of motifs on the 5' external transcribed spacer, upstream from the primary pre-rRNA processing site in a higher plant.

In radish the primary processing site in pre-rRNA has been mapped to a TTTTCGCGC sequence (motif P) in the 5' external transcribed spacer (5' ETS) of the ribosomal DNA (rDNA) [Delcasso-Tremousaygue, D., Grellet, F., Panabières, F., Ananiev, E. & Delseny, M. (1988) Eur. J. Biochem. 172, 767-776]. The processing site is just downstream of four similar motifs named A1, A2, A3 and B. The five motifs constitute cluster A123BP. We have described previously that in radish extracts a nuclear protein, nuclear factor B (NF B) specifically binds to motif B [Echeverría, M., Penon, P. & Delseny, M. (1994) Mol. Gen. Genet. 243, 442-452]. Here, by means of electrophoretic-mobility-shift assays, we describe an rDNA-binding activity, nuclear factor D (NF D), that interacts with the A123BP cluster. Using various rDNA probes and competitors we show that NF D binds specifically to the A123 clustered motifs but not to similar B or P motifs. We used sequence-specific DNA-affinity chromatography to separate NF D from NF B. DNase I footprinting was used to map the binding site of NF D on the A123BP cluster and we compared it with that of NF B on the same probe. The footprint of NF D extends from the A1 motif to the 5' end of the NF B-binding site and includes motifs A2 and A3 on each strand. The footprinting of NF B is restricted to motif B and adjacent nucleotides. Thus the NF D-binding and NF B-binding sites are distinct but overlap. These two factors bind with a high specificity to the A123BP cluster in the radish 5' ETS. The possibility that these factors regulate rDNA transcription elongation at the level of the primary pre-rRNA processing site in crucifers is discussed.

Functional study of multidrug resistance with fluorescent dyes. Limits of the assay for low levels of resistance and application in clinical samples.

Fluorescent dyes such as rhodamine 123 (R123) and Hoechst 33342 (Ho342) have been widely used to characterize multidrug-resistance (MDR) phenotype cells in cell populations, on the basis of their reduced accumulation in resistant cells. Taking advantage of the high fluorescence quantum yield of R123 and Ho342 compared with that of anthracyclines, we investigated the limits of fluorescence image cytometry in detecting MDR by the level of R123 and Ho342 accumulation and efflux. We were able to separate with this technique a cell line with a level of resistance as low as 3. We then studied the presence of MDR cells in lymphocytes isolated from patients with hematological malignancies.

Identification of a 67 kDa protein that binds specifically to the pre-rRNA primary processing site in a higher plant.

In radish pre-rRNA primary processing cleavage occurs at a UUUUCGCGC element (motif P) mapped in the 5'-external transcribed spacer (Delcasso-Tremousaygue et al., 1988). Significantly, motif P is part of a cluster of homologous elements including three UUUUCCGG elements (motifs A123) and a single UUUUGCCCC element (motif B). Here we used the EMSA to identify in radish extracts an RNA-binding activity, NF C, that specifically interacts with the pre-rRNA A123BP sequence. Using different RNA probes and competitors we show that NF C recognises a 38 base RNA sequence including the 3'-end of motif A3 and motifs B and P. NF C binds to poly U, but not to poly A, poly C or poly G. Therefore we used poly (U) Sepharose chromatography as a final step to obtain pure NF C fractions. These, analysed by SDS-PAGE, revealed two major polypeptides of 67 and 60 kDa. According to UV cross-linking analysis the 67 kDa polypeptide corresponds to NF C activity, while the 60 kDa species is a proteolysed form of this protein. We also showed that NF C is enriched in nuclear extracts. Based on its stringent RNA substrate specificity and its nuclear localisation we propose that NF C is involved in pre-rRNA primary processing in plants.

Binding to DNA and cytotoxic evaluation of ascididemin, the major alkaloid from the Mediterranean ascidian Cystodytes dellechiajei.

The isolation of ascididemin from the Mediterranean ascidian Cystodytes dellechiajei is described. This alkaloid consists of a planar pentacyclic chromophore which was investigated for its DNA-binding and cytotoxic properties. Spectroscopic measurements provided evidence that the drug intercalates into DNA. DNase I footprinting assays indicated that the binding of ascididemin to GC-rich sequences is favoured over binding to AT-rich and mixed sequences. Chemical probes were used to detect ligand-induced structural changes in DNA. The alkaloid induces a hyper-reactivity of the DNA towards potassium permanganate, but not towards diethylpyrocarbonate, just as is the case with ethidium bromide; it has little effect on the catalytic activities of topoisomerases I and II. Ascididemin exhibits marked cytotoxicity towards human leukaemic cells in vitro and appears to be practically equally toxic for drug-sensitive and multidrug-resistant cell lines. The results suggest that DNA, but not topoisomerases, may represent the critical cellular target at which this marine alkaloid exhibits its potent cytotoxic properties in vitro.

Is reduced accumulation of Hoechst 33342 in multidrug resistant cells related to P-glycoprotein activity?

Although bisbenzimidazole-DNA interactions have been studied in solution, little information has been available in living cells. The reduced accumulation of the nuclear dye Hoechst 33342 (H342) in cells with multidrug resistant (MDR) phenotype suggested its possible use in a functional test for detection of these cells. We performed experiments to elucidate the mechanisms involved in the H342-exclusion from resistant cells. As contradictory results have been reported in literature, we compared the entire fluorescence spectra of H342 in solution and in intact living cells under different experimental conditions. The study was performed by fluorescence image cytometry. This technique allow accurate quantification of the amount of H342 bound to DNA in living cells. The dye uptake was followed in sensitive and resistant cells, a lymphoblastoid cell line, CCRF-CEM, and its resistant variant selected with vinblastine CEM/VLB100 under conditions that could modulate H342-cell binding. Competition experiments with sodium azide, verapamil, and vinblastine indicated that resistant cells did not differ in the number of possible binding sites for H342. The obtained results ruled out the possibility of discriminating cells on the basis of a spectral shift. Two modes of binding, differing in their affinity for the dye, seem to co-exist in intact cells. Although it clearly appeared that the P-glycoprotein expressed in MDR cells was mainly responsible for the H342-exclusion, other mechanisms might also be involved.

Staining with Hoechst 33342 and rhodamine 123: an attempt to detect multidrug resistant phenotype cells in leukemia.

Development of resistance is the major cause of failure in chemotherapeutic treatments. We have previously shown that the level of labeling with Hoechst 33342 and rhodamine 123 in established cell lines was decreased in cells with 'classic' MDR phenotype. This functional test was carried out using fluorescence image cytometry on living cells. We applied this protocol to patients with chronic lymphocytic leukemia. Although a large variability of the labeling is observed in cells from healthy donors, this approach seems to be useful for early detection of P-gp-dependent resistance in leukemia cells and for identification of new reversing agents on patient lymphocytes.

Nile red labeling of single living cells for contour delineation to quantify and evaluate the distribution of rhodamine 123 with fluorescence image cytometry.

Simultaneous study of intracellular quantification and distribution of fluorescent probes is difficult when cell staining is not homogeneous. This occurs after mitochondrial staining with rhodamine 123 (R123). Classical techniques for evaluation of intracellular R123 fluorescence, such as flow cytometry, are based on measurement of the global fluorescence intensity but do not take into account parameters that reflecting cellular distribution of the probe. For simultaneously studying intracellular quantification and distribution of R123 with fluorescence image analysis, we delineated a mask of the cell, generated from a fluorescent image of the plasma membrane stained by nile red (NR). After a preliminary study of the fluorescence characteristics of R123 and NR to avoid artifacts and optimize conditions of staining, quantification and distribution of intracellular R123 studies were performed by superimposition of the mask on the R123 fluorescence image. This protocol was applied to leukemic cells and allowed estimation of individual cell parameters such as mean fluorescence intensity and standard deviation, the latter providing information of the cellular distribution of R123. Moreover, it permitted demonstration of the redistribution of R123 in the whole cell when coincubated in the presence of nigericin.

Detection of human leukemia cells with multidrug-resistance phenotype using multilabeling with fluorescent dyes.

Reduced accumulation of multiple drugs is a characteristic of cells overexpressing P-glycoprotein. This phenotype is referred to as multidrug-resistance (MDR). A protocol based on reduced accumulation of fluorescent dyes is proposed for discriminating MDR cells in cell populations. The combination of three fluorescent dyes, Hoechst 33342, rhodamine 123 and Nile red, with different intracellular targets, has been designed to characterize cells with different levels of resistance, using image cytometry. The fluorescence intensity of each dye was quantified in living cells. The protocol was applied to human leukemia cell lines, (K562, K562/ADR, CCRF-CEM, CEM/VLB100, CEM/VM-1). The effect of verapamil on dye accumulation is emphasized.

Identification of multi-drug resistant cells in sensitive Friend leukemia cells by quantitative videomicrofluorimetry.

The cellular resistance to cytotoxic drugs, particularly to anthracyclines, remains a major problem in cancer chemotherapy. A number of biochemical mechanisms have been described, one of them being a lower accumulation of drugs in resistant cells. The accumulation of Ho33342 in sensitive and resistant Friend leukemia cells was studied by quantitative fluorescence image analysis, a method which allows investigations to be made on living tissues and cells. The intensity of fluorescence is related to the amount of Ho33342 accumulated into the cells and has been found to be more intense in sensitive cells than in resistant ones. Moreover, the retention of this vital dye was inversely related to the degree of resistance in the three resistant cell lines. The addition of verapamil, which is known to reverse resistance to anthracyclines, resulted in an increase of the amount of Ho33342 accumulated in the resistant cells. Ho33342 presents a higher quantum yield than any other anthracyclines, such as adriamycin and can be used as a microfluorimetric probe to identify the resistant cells in a heterogeneous cell population.

Fluorescent image cytometry: from qualitative to quantitative measurements.

Image analysis is being increasingly used in biology and medicine; however, in order to obtain truly quantitative data and thus avoid errors in interpretation, a certain number of precautions must be taken when the image is digitized, well before any attempt is made to analyse or interpret the data. This is particularly true for image microfluorometry. In this article we will examine an image analysis system for fluorescent images composed of a mercury lamp, a microscope, a high sensitivity video camera and an image analyser and evaluate the principal sources of random and non-random errors, various constraints, and their relative importance. A signal correction protocol is proposed to minimize non-random errors during digitalization. A few examples are given to illustrate its efficiency.

pHi and DNA content modifications after ADR treatment in 3T3 fibroblasts. A microfluorimetric approach.

This paper attempts to correlate morphological changes observed after ADR treatment with other cellular parameters. Experiments were carried out on control cells, ADR and cysteamine treated cells and the evaluation of DNA content and the determination of the pHi were studied using microspectrofluorimetry and Numerical Image Analysis. Results show that the DNA content and the pHi are modified significantly in presence of ADR but that no changes are observed when cells are treated with cysteamine alone. Furthermore no correlation could be established between these parameters and the class of cells that does not accumulate BaP. Further investigation will be necessary, first on other parameters such as intracellular Ca++, and second, by following parameters simultaneously on the same cell.

MFO activity changes in single living 3T3 fibroblasts treated with adriamycin.

The effects of Adriamycin on 3T3 fibroblasts has been followed by microspectrofluorimetry. MFO activity was monitored by BaP metabolism and three points were studied: long-term ADR treatment, reversibility of this treatment and cysteamine effect on ADR treatment. The appearance of a sub-population that does not accumulate BaP suggests either a greater efflux of BaP or no penetration of the compound after ADR treatment. Moreover drastic morphological changes were observed.

Influence of long term treatment with 3-methylcholanthrene or carbaryl on mixed-function oxidase activity in 3T3 fibroblasts: studies by microspectrofluorimetry on single cells.

Using benzo(a)pyrene (BaP) as a probe for aryl hydrocarbon hydroxylase (AHH) activity, differences in mixed-function oxidase (MFO) activity were observed using microspectrofluorimetry in single living cells during long term treatment with 3-methylcholanthrene (3-MC) or carbaryl. Although these two compounds differ in chemical structure, similar effects were observed in 3T3 cell populations. The results suggest that the two compounds activate the same enzymatic system and that individual cells of a supposed homogeneous cell population are not equally sensitive to xenobiotics, i.e. subpopulations were observed which have differences in AHH activity.

Effect of ellipticine on MFO activity in single living 3T3 fibroblasts studied by microspectrofluorimetry.

The MFO activity in intact cells was monitored through microspectrofluorimetry study of BaP metabolization. This approach allowed observation of individual cells in a cell population to represent the studied samples through histograms as a function either of rate constants or of "residual BaP". The inhibitory effect of ellipticine was demonstrated on intact living 3T3 fibroblasts. The role of DMF dissolution and cellular localisation of compounds were also studied.

Microspectrofluorometric comparison of benzo(a)pyrene and dibenzo(c,h)acridine metabolism in single living 3T3 fibroblasts.

The purpose of this study was to determine if dibenzo(c,h)acridine could be used as a probe for the N-heterocyclic aromatic compound detoxification system. The determination was achieved by cell population studies (histograms) using microspectrofluorimetry on single living cells. The results show a similar metabolic distribution pattern between dibenzo(c,h)acridine and benzo(a)pyrene, whereas important differences appear between 6-aminochrysene and dibenzo(c,h)acridine, respectively amine aromatic and N-heterocyclic aromatic compounds. No differences were observed in the metabolism of benzo(a)pyrene in untreated cells and those treated with constant concentrations of dibenzo(c,h)acridine. However, inhibition of dibenzo(c,h)acridine metabolism was observed in the presence of benzo(a)pyrene, indicating that this compound should be a preferential substrate for a cell detoxification system.

[Demonstration of the intracellular accumulation of intermediary fluorescent metabolites of various polycyclic aromatic hydrocarbons by resolution of the fluorescence spectra of isolated living cells]. / Mise en évidence de l'accumulation intracellulaire de métabolites intermédiaires fluorescents de quelques hydrocarbures aromatiques polycycliques par résolution de spectres de fluorescence de cellules vivantes isolées.

Using microspectrofluorimetry, we have studied some spectral modifications induced by the accumulation of fluorescent metabolites from some PAH in single living cells (3T3 and RTG2 fibroblasts). Whatever the parent compound under study, no spectral deformation has been detected in the 3T3 cells. On the contrary, the characteristic fluorescence spectra were strongly modified in RTG2 cells when the parent compound was either B(a)P or CPAP. The B(a)P metabolites have been identified as 3-OH-B(a)P and 9-OH-B(a)P.

Quantitative microspectrofluorimetry study of the "blocking effect" of 6-aminochrysene on benzo[a]pyrene metabolism using single living cells.

By microspectrofluorimetry on single living cells (murine fibroblasts 3T3), we have obtained monoexponential decreases of fluorescence intensity for benzo[a]pyrene (B[a]P) and 6-aminochrysene (6a-chrysene) metabolism. These kinetics are characteristics of B[a]P and 6a-chrysene metabolism and histograms can be drawn from the rate constants. We have studied the influence of 6a-chrysene on B[alpha]P metabolism. Using different methods of incubation, it has been observed that the presence of 6a-chrysene leads to modifications of the histogram profiles during B[a]P metabolism. Polycyclic aromatic hydrocarbons (PAH) are used to induce B[a]P metabolism. Whatever the experimental conditions we never detected such a phenomenon with 6a-chrysene. On the contrary we have observed an inhibition of B[a]P metabolism by 6a-chrysene, which can reach 80% of the aryl hydrocarbon hydroxylase (AHH) activity when 6a-chrysene remains constant in the cells. Compared with the results previously observed "in vitro" which presented 50% mean inhibition) we show that inhibition acts in an all-or-nothing mechanism at the cellular level.

[Fluorescence decay curves under pulsed excitation: distinction between free and bound NAD(P)H on isolated living cells]. / Courbes de déclin de la fluorescence excitée en régime pulsé: distinction du NAD(P)H libre et du NAD(P)H lié sur des cellules vivantes isolées.

Under pulsed excitation the decay curve of the intrinsic cellular fluorescence has been recorded using an original equipment. Cell by cell experiments are feasible so that the NAD(P)H bound/free ratio and the relaxation time characteristic of bound NAD(P)H can be used in order to build up histograms characterizing the cell population.