Whole-mount in situ detection of microRNAs on Arabidopsis tissues using Zip Nucleic Acid probes.

MicroRNAs (miRNAs) affect fundamental processes of development. In plants miRNAs regulate organ development, transition to flowering, and responses to abiotic/biotic stresses. To understand the biological role of miRNAs, in addition to identifying their targeted transcripts, it is necessary to characterize the spatiotemporal regulation of their expression. Many methods have been used to define the set of organ-specific miRNAs by tissue dissection and miRNA profiling but none of them can describe their tissue and cellular distribution at the high resolution provided by in situ hybridization (ISH). This article describes the setup and optimization of a whole-mount ISH protocol to target endogenous miRNAs on intact Arabidopsis seedlings using DIG-labeled Zip Nucleic Acid (ZNA) oligonucleotide probes. Automation of the main steps of the procedure by robotized liquid handling has also been implemented in the protocol for best reproducibility of results, enabling running of ISH experiments at high throughput.

Sequence specific peptidomimetic molecules inhibitors of a protein-protein interaction at the helix 1 level of c-Myc.

Our work is focused in the broad area of strategies and efforts to inhibit protein-protein interactions. The possible strategies in this field are definitely much more varied than in the case of ATP-pocket inhibitors. In our previous work (10), we reported that a retro-inverso (RI) form of Helix1 (H1) of c-Myc, linked to an RI-internalization sequence arising from the third alpha-helix of Antennapedia (Int) was endowed with an antiproliferative and proapoptotic activity toward the cancer cell lines MCF-7 and HCT-116. The activity apparently was dependent upon the presence of the Myc motif. In this work, by ala-scan mapping of the H1 portion of our molecules with D-aa, we found two amino acids necessary for antiproliferative activity: D-Lys in 4 and D-Arg in 5 (numbers refer to L-forms). In the natural hetero-dimer, these two side chains project to the outside of the four alpha-helix bundle. Moreover, we were able to obtain three peptides more active than the original lead. They strongly reduced cell proliferation and survival (RI-Int-VV-H1-E2A,S6A,F8A; RI-Int-VV-H1-S6A,F8A,R11A; RI-Int-VV-H1-S6A,F8A,Q13A): after 8 days at 10 muM total cell number was approximately 1% of the number of cells initially seeded. In these more potent molecules, the ablated side chains project to the inside in the corresponding natural four alpha-helix bundle. In the present work, we also investigated the behavior of our molecules at the biochemical level. Using both a circular dichroism (CD) and a fluorescence anisotropy approach, we noted that side chains projecting at the interior of the four alpha-helix bundle are needed for inducing the partial unfolding of Myc-H2, without an opening of the leucine zipper. Side chains projecting at the outside are not required for this biochemical effect. However, antiproliferative activity had the opposite requirements: side chains projecting at the outside of the bundle were essential, and, on the contrary, ablation of one side chain at a time projecting at the inside increased rather than decreased biological activity. We conclude that our active molecules probably interfere at the level of a protein-protein interaction between Myc-Max and a third protein of the transcription complex. Finally, CD and nuclear magnetic resonance (NMR) data, plus dynamic simulations, suggest a prevalent random coil conformation of the H1 portion of our molecules, at least in diluted solutions. The introduction of a kink (substitution with proline in positions 5 or 7) led to an important reduction of biological activity. We have also synthesized a longer peptido-mimetic molecule (RI-Int-H1-S6A,F8A-loop-H2) with the intent of obtaining a wider zone of interaction and a stronger interference at the level of the higher-order structure (enhanceosome). RI-Int-H1-S6A,F8A-loop-H2 was less active rather than more active in respect to RI-Int-VV-H1-S6A,F8A, apparently because it has a clear bent to form a beta-sheet (CD and NMR data).

HPV16-E6 enhances mitoxantrone sensitivity in a human ovarian cancer line: an isolated instance or a trend?

A cellular isogenic system, in which wt-p53 expression level is challenged through human papilloma virus 16-E6 gene transfection, was previously developed in our laboratory. As an average trend, cancer lines bearing an inactivated p53 have a general tendency toward an increased resistance to chemotherapeutic agents. However, using the above isogenic system, the transfected line (A2780-N9) was found to be more sensitive to taxol than the parental one (A2780-WT). In a NCI meta-analysis study the average trend is that altered p53 status is related to cellular resistance to topoisomerase II inhibitors, while it is irrelevant in determining sensitivity/resistance to mitotic spindle poisons. We report that our E6 transfected line, previously shown to be hypersensitive to taxol, is also clearly hypersensitive to a topoisomerase II inhibitor (mitoxantrone). Differences in cytotoxicity are more evident after a shorter/more intense exposure, than after a milder/longer exposure, being A2780-WT 27-fold more resistant than the transfected clone in the former case. These differences seem to be related to the different activities ("cross-talks") of E6 protein, among which shortening of p53 half-life is only one aspect. After mitoxantrone treatment A2780-N9 cells display also an increased propensity to apoptosis. In addition, a literature survey of E6 effects in transfected cancer cell lines, seems to suggest that chemosensitization to different classes of antineoplastic agents is the rule rather than the exception in these E6-based isogenic systems.

Biological effects of a neurotoxic pesticide at low concentrations on sea urchin early development. A terathogenic assay.

Dose-dependent terathogenic effects of an organophosphate insecticide were found during early sea urchin development. This biological assay is low cost, easy to measure, and allows to detect the effects of the exposure of organisms to the active principle at concentrations lower than the acceptable daily intake (ADI) for man. Effects were found independently from the stage of exposure, and were major as earlier exposure occurred. The stronger effects were exerted on the elongation of the skeletal rods, that was easily measured by following the migration of primary mesenchyme cells, labelled by WGA (wheat germ agglutinin).

Bcl-xL modulates apoptosis induced by anticancer drugs and delays DEVDase and DNA fragmentation-promoting activities.

Using an episomal eucaryotic expression vector, we derived three stable transfected human leukemic U-937 variant lines showing differential expression of the Bcl-xL protein. Preventive effect of Bcl-xL on cell death induced by various concentrations of camptothecin (DNA topoisomerase I inhibitor; CPT) was observed in the three lines with most pronounced effect in cells containing the highest level of Bcl-xL expression. These results show that increased cell death protection by Bcl-xL is correlated with its level of expression. The extent of DNA strand break formation and DNA synthesis inhibition following CPT treatments was similar in control and transfected U-937 cells, suggesting that Bcl-xL acts downstream of CPT-DNA topoisomerase I-mediated DNA strand breaks. Modulation of cell death by Bcl-xL was also observed in cells treated with etoposide, vinblastine, paclitaxel, and cisplatinum (II) diammine dichloride. To define whether Bcl-xL functions downstream or upstream of apoptogenic proteolytic cascade activation, we compared kinetics of DNA fragmentation in treated cells with kinetics of caspase 1-like, caspase 3-like, and N-tosyl-L-phenylalanylchloromethyl ketone (TPCK)-sensitive activities. In CPT-treated U-937 cells, caspase 3-like and TPCK-sensitive activities promoting DNA fragmentation in a cell-free system were detected much more rapidly in extracts obtained from CPT-treated U-937 cells compared to those obtained from CPT-treated U-937-Bcl-xL variant cells. These results suggest that Bcl-xL delays their activation that correlates with the occurrence of DNA fragmentation. Addition of recombinant Bcl-xL in extracts containing DEVDase and TPCK-sensitive activities did not inhibit these activities, suggesting that Bcl-xL acts primarily upstream of their activation in the apoptotic process. Taken together, these results suggest that Bcl-xL is a primary checkpoint that can block or delay transmission of cell death signals emerging from DNA damage and prevents activation of an apoptogenic proteolytic cascade.

Muscarinic drugs affect cholinesterase activity and development of eye structures during early chick development.

During neurogenesis, markers of the cholinergic system are present in the eye and visual cortex of vertebrates. In adult vertebrates, a role for these molecules, including muscarinic acetylcholine receptors (mAChRs), in eye growth non-accommodative regulation is also known. In order to understand the biological mechanisms triggered by the cholinergic system in these events, we analysed the effects of a cholinergic agonist (10(-4) M carbachol) and an antagonist (10(-4) M atropine) of the muscarinic receptors, on early chick development. To establish if the cholinergic system also plays a role in the regulation of early neurogenetic signals, the drug treatments were made at stage 5-6 HH, during the formation of the cephalic process. Specific effects on forehead, and in particular on eye development were found; carbachol treated embryos presented huge and well pigmented eyes, significantly different from controls. The eyes of atropine-exposed embryos presented anomalies with different phenotypes ranging from strongly affected features to normal-like appearance. Generally, the eyes were smaller as compared to the controls, with a number of anomalies, also in the normal-like phenotype, including retina and lens defects. In these structures, distribution of cholinesterase activities was checked by histochemical methods, and the amount of cells undergoing nuclear disgregation was revealed by DAPI staining. We propose that the drugs affected the known nervous and pre-nervous functions of the cholinergic markers, such as cell signalling during primary induction, and regulation of cell death by ACh receptors.

Bax-alpha promotes apoptosis induced by cancer chemotherapy and accelerates the activation of caspase 3-like cysteine proteases in p53 double mutant B lymphoma Namalwa cells.

Stable transfected human p53 (mt/mt) B lymphoma Namalwa variant lines showing differential expression of the Bax-alpha protein were derived under hygromycin selection. Overexpression of Bax-alpha in these variant cells accelerates cell death induced by short or continuous treatments with various concentrations of camptothecin, etoposide, vinblastine and shows no accelerating cell death activity in cis-platinum and paclitaxel-treated cells. Activation of apoptosis and oligonucleosome-sized DNA fragmentation was observed in the variant lines with more pronounced effect in cells containing high level of Bax-alpha protein. These results suggest that increased cell death mediated by anticancer drugs correlates with Bax-alpha level of expression and that Bax-alpha sensitizes Namalwa cells treated at low drug concentrations. The extent of DNA synthesis inhibition following DNA topoisomerase inhibitor treatments was similar in control and all transfected Namalwa cells suggesting that Bax-alpha acts downstream of DNA topoisomerase-mediated DNA strand breaks. To define further the relation between Bax-alpha expression and apoptosis activation, kinetics of caspase activation was measured in drug-treated cells. Caspase activities were measured using specific fluorogenic peptide derivatives DABCYL-YVADAPV-EDANS and Ac-DEVD-AMC, substrates of the caspase 1-like and caspase 3-like families, respectively. In control and Bax-alpha transfected Namalwa cells no increase in caspase 1-like activity was detected following camptothecin and etoposide treatments. In contrast, a significant difference in Ac-DEVD-AMC hydrolysis activity was observed in Bax-alpha transfected Namalwa cells compared to that of control Namalwa cells after camptothecin and etoposide treatment. Increased caspase 3-like activity correlated also with poly(ADPribosyl) polymerase cleavage. Taken together, these results suggest that Bax-alpha sensitize B lymphoma cells to series of anticancer drugs and accelerates the activation of apoptotic protease cascade.

The Bcl-xL and Bax-alpha control points: modulation of apoptosis induced by cancer chemotherapy and relation to TPCK-sensitive protease and caspase activation.

Defective control of apoptosis appears to play a central role in the pathogenesis of human diseases including neoplasic, autoimmune, and neurodegenerative diseases. Conversely, cancer chemotherapy and ionizing radiation can induce cancer cell death by apoptosis, and deregulated apoptosis following cancer chemotherapy could define a new category of drug resistance mechanism. By understanding the role that some major regulators of apoptosis play either at the commitment or execution phases of cell death in a given tissue and pathology, we will be in a better position to design and explore new therapeutic modalities. The Ced-9 - Bcl-like and Ced-3 - Ice-like gene family products are intrinsic proteins regulating the decision of a cell to survive or die and executing part of the cell death process itself, respectively. Among the various Bcl-like proteins, the effects and functions of the Bcl-x and Bax proteins in controlling apoptosis induced by cancer chemotherapy have been studied recently. In human cancer variant cell lines showing differential expression of the Bcl-xL protein, a preventive effect of Bcl-xL on cell death induced by various cytotoxic drugs is observed, with greater effects in cells containing the highest level of Bcl-xL expression. Similarly, overexpression of Bax-alpha in cancer cell lines sensitizes these cells to some cancer chemotherapy compounds. Modulation of apoptosis either negatively by Bcl-xL or positively by Bax-alpha resides downstream of the primary mechanism of action of anticancer drugs, suggesting that they act primarily as intrinsic control points following cytotoxic drug injuries. An emerging family of Ced-3 - Ice like cysteine proteases (caspases) has been also identified and several studies have revealed their importance in executing the process of cell death. More recently, activation of a N-tosyl-L-phenylalanylchloromethyl ketone (TPCK)-sensitive pathway was also suggested to play an important role in apoptosis induction following cancer chemotherapy. Evidence obtained using a combination of assays including cell-free systems and enzyme activity assays now suggests that Bcl-xL and Bax-alpha control points function upstream of TPCK-sensitive protease and caspase activation. Bcl-xL delays and prevents activation of apoptotic protease cascades whereas Bax-alpha shows the opposite effect, accelerating their activation.

Interactions between taxol and camptothecin.

Taxol is an antitumor drug which, as its mechanism of action, promotes microtubule assembly in vitro. Camptothecin (CPT) is an anticancer agent with the peculiar mechanism of poisoning eukaryotic DNA topoisomerase I. Both drugs are in clinical trials and their chemotherapeutic efficacy seems promising in refractory human ovarian cancer. We studied the molecular and cellular pharmacology of the two drugs when administered simultaneously toward human ovarian cancer cell line A2780. Taxol inhibits CPT-induced single-strand breaks as well as CPT-induced cytotoxicity. Taken together, our experiments indicate that the two drugs might interact with the same class of nuclear enzyme, i.e. DNA topoisomerase I.

Tumour necrosis factor enhances the therapeutic effect of mitoxantrone in human ovarian cancer xenograft.

The combination of Tumour Necrosis Factor (TNF) and mitoxantrone was evaluated for potential chemotherapeutic effect against a human ovarian cancer cell line A2774 hetero-transplanted in female nude mice. Both antitumour efficacy (relative survival and reduction of ascites) and toxicity (weight loss and liver toxicity) of TNF alone, mitoxantrone alone or TNF + mitoxantrone were evaluated. A significant difference (P < 0.002) was observed only among animals bearing tumours treated with mitoxantrone (0.012 mg/Kg) + TNF (5 x 10(5) U/Kg) and controls. No cytotoxic effects were observed for this combination. These observations provide a rationale for further evaluation of TNF + mitoxantrone based regimes for the treatment of recurrent ovarian cancer.

Interferon-gamma enhances TNF sensitivity in A172 human glioblastoma cell line.

Although human glioblastoma cell line A172 presents tumor necrosis factor (TNF) high affinity receptors, it is resistant to TNF-mediated cytotoxicity. Preincubation of cells with 10 ng/ml interferon (IFN)-gamma for 6 h causes a great increase in TNF receptor numbers (about 477%). IFN-gamma alone is not cytotoxic in A172 cells. Cells preincubated with 10 ng/ml IFN-gamma for 6 h became more sensitive to the TNF cytotoxicity (about 400%). In A172 cells the enhanced TNF receptor expression may contribute to the enhancement of TNF cytotoxicity. This observation may have importance in designing new more effective therapies for human gliomas.

Interferon-alpha or beta potentiate platinum analogous in human glioblastoma cell lines.

The effect of interferon-alpha or beta on platinum analogues [cisplatin (CDDP) and carboplatin] cytotoxicity was studied in four glioblastoma cell lines (U373MG, T98G, A172 and U118Mg). All cell lines were strongly resistant to the cytotoxic effect of CDDP or carboplatin. Although both interferons were not cytotoxic in all cell lines, they were able to significantly increase the cell platinum-sensitivity. Specifically interferon-alpha increased the magnitude of CDDP-induced DNA interstrand crosslinks. Our findings suggest that interferons are able to induce a very strong potentiation of platinum analogues cytotoxicity in drug-resistant human glioma cell lines.

Sensitization of human glioblastoma T98G cells to VP16 and VM26 by human tumor necrosis factor.

The effect of Tumor Necrosis Factor (TNF) on VP16 or VM26 cytotoxicity was studied in a human glioblastoma cell line T98G, which expresses TNF-receptors. Although T98G cells did not produce TNF endogenously they were resistant to the cytolytic effect of TNF. T98G cells were also moderately sensitive to the action of VP16 or VM26. TNF given at 1000 U/ml was able to increase the cell drug-sensitivity significantly. This effect was due to an increase in the VP 16-induced cleavable-complexes by TNF. These findings suggest that TNF specifically sensitizes human glioma T98G cells to the effects of VP 16 of VM26.

"Atypical" multidrug resistance in human ovarian cancer cell line A2780 selected for resistance to doxorubicin (A2780 DX3).

Human ovarian cancer cells A2780, selected for resistance to doxorubicin (A2780-DX3), are cross-resistant to various other topoisomerase-II-targeted drugs but not to vinblastine. The parental cell line was very sensitive to doxorubicin-, mitoxantrone- or etoposide(VP16)-induced DNA single-strand breaks, under deproteinizing conditions. In contrast, little or no DNA strand breakage was seen in resistant A2780-DX3 cells, even at very high concentrations, indicating a good correlation, with cytotoxicity. No significant alterations in cellular drug uptake were observed in DX3 cells. Further studies showed that the nuclei isolated from resistant cells were also resistant to mitoxantrone- or VP16-induced single-strand breaks, indicating that nuclear modifications in resistant cells are responsible for this resistance. Catalytic activity in crude nuclear extracts from wild-type and DX3 cells was almost equal. However, an assay that specifically measures generation of 5'-protein-linked breaks in 32P-labeled 3 DNA revealed that, DNA cleavage activity in nuclear extract from the DX3 cell line is profoundly resistant to a stimulation by VP16. These data indicate that stimulation of topoisomerase-II-mediated DNA cleavage is responsible for topoisomerase-II-targeted drug-cytotoxicity rather than loss of normal topoisomerase catalytic function. These data support the hypothesis that A2780-DX3 cells display an "atypical" multidrug resistance.

Tumor-necrosis-factor and DNA topoisomerase-ii inhibitors in human ovarian-cancer - potential role in chemotherapy.

Seven ovarian and one cervical human cancer cell lines were examined far their sensitivity or resistance to tumor necrosis factor, to three topoisomerase II inhibitors and to cisplatin. Only one line exhibited the multidrug-resistance phenotype and another one an 'atypical'-MDR phenotype. The combination of TNF and topoisomerase-II inhibitors produced enhanced cytotoxicity and overcame the MDR and the atypical resistance. No potentiation of cisplatin cytotoxicity was observed. These findings suggest that TNF enhances the activity of DNA topoisomerase II both in TNF resistant and sensitive cells.

Sister-chromatid exchanges, chromosomal aberrations and cytotoxicity produced by topoisomerase II-targeted drugs in sensitive (A2780) and resistant (A2780-DX3) human ovarian cancer cells: correlations with the formation of DNA double-strand breaks.

Doxorubicin, ellipticine and etoposide are antineoplastic drugs with topoisomerase II inhibitory activity. The relationship between drug-induced sister-chromatid exchanges (SCEs) or chromosomal aberrations (CAs) and cytotoxicity, or drug-induced DNA double-strand breaks (DSBs) and cytotoxicity, or drug-induced SCEs and DSBs was investigated in human ovarian cancer cells sensitive (A2780) and resistant (A2780-DX3) to topoisomerase II inhibitors. 30-min drug treatments produced SCEs, CAs and DSBs in sensitive cells, doxorubicin being more potent than etoposide at equimolar concentrations. The same treatments of resistant (A2780-DX3) cells did not produce chromosomal damage (SCEs, CAs, DSBs) and no cytotoxicity was observed. A plot of cytotoxicity versus SCEs indicated a good correlation between these two parameters for topoisomerase II inhibitors and not for mytomicin C. The plot of DSBs versus SCEs also showed a very good correlation.

Induction of DNA double-strand breaks by 8-methoxycaffeine: cell cycle dependence and comparison with topoisomerase II inhibitors.

We have studied the ability of 8-methoxycaffeine (8-MOC)--one of the most effective caffeine derivatives in inducing chromosomal aberrations--to induce DNA double strand breaks (DSB) in purified human T lymphocytes during the cell cycle. Etoposide- or ellipticine-mediated DNA break frequency was used as a parameter of topoisomerase II activity. DNA-DSB induced by either 8-MOC or VP16 or ellipticine rose co-ordinately with the level of DNA topoisomerase II and with the onset of DNA replication. At concentrations between 10 and 50 microM 8-MOC was approximately 75% as active in terms of DSB as VP16 and ellipticine. By contrast with VP16 and ellipticine, 8-MOC was not cytotoxic. In conclusion, our data suggest that 8-MOC is an agent that efficiently induces DNA-DSB at non-toxic concentrations, and without direct inhibition of topoisomerase II.

Circumvention of atypical multidrug resistance with tumor necrosis factor.

Some "multidrug-resistant" (MDR) cell lines are not associated with a defect in drug accumulation or with the overexpression of P-glycoprotein. These cell lines are defined as "atypical MDR" (at-MDR) and they often express altered or mutated topoisomerase II. We investigated the ability of tumor necrosis factor to reverse at-MDR (in the human ovarian cancer cell line A2780 DX3) on the basis of its efficacy in potentiating in vitro topoisomerase II-targeted drugs, and because there is convincing evidence that the synergy is due to an increased number of topoisomerase-associated strand-breaks as well as to an increased level of extractable topoisomerase.

Potentiation of TNF-mediated cell killing by mitoxantrone. Relationship to DNA single-strand break formation.

Tumor necrosis factor (TNF) is a pleiotropic cytokine that mediates different cellular responses including cytotoxicity, cytostasis, proliferation, differentiation and expression of specific genes. Recent studies have demonstrated that chemotherapeutic drugs that inhibit the nuclear enzyme DNA topoisomerase II synergize with TNF in tumor cell killing in vitro and in vivo. We now report that a combination of TNF and the topoisomerase II inhibitor Mitoxantrone produced dose-dependent synergistic cytotoxicity against the human ovarian cancer cell line A2774 in a clonogenic assay (1 hr treatment). This result was obtained with simultaneous administration of the drug and the cytokine under test, and is independent of modification of Mitoxantrone uptake. This combination is responsible for an evident augmentation of "cleavable complex" formation. From isolated nuclei, we have isolated also the topoisomerase II activity; we observed an increment when the cells were previously treated with TNF, 2.5 min before nuclear extraction. After 10-30 min of treatment with TNF, the topoisomerase II activity returned to normal values. If TNF is not given with but 30 min before Mitoxantrone, no potentiation of cytotoxicity or break induction is observed. These results suggest that specific timing of the association may be needed also when attempting to translate it to animals and humans.