Biomedical subjects

R J Heetebrij

Publications and source records attributed to R J Heetebrij.

6 recordsLinked to original sources

PubMed2003

Platinum(II)-based coordination compounds as nucleic acid labeling reagents: synthesis, reactivity, and applications in hybridization assays.

The synthesis, characterization, and molecular interactions of platinum(II) coordination compounds, which contain a distal nonradioactive reporter molecule, with mono- and polynucleotides are described. A [Pt(II)(en)(NH(2)(CH(2))(6)NH-tBoc)Cl](NO(3)) (en=ethylenediamine) entity has been coupled, after removal of the tBoc group, to a number of hapten and fluorophore molecules through succinimide derivatives. The influence of the various tethered reporter groups within these complexes on the reactivity towards guanosine 5'-monophosphate (5'-GMP), as a model for polynucleotide sequences, was investigated to shed light on the use of these reagents in hybridization assays. Reactivity turned out to be strongly dictated by the chemical nature of the distal reporter molecule present. At pH 7.0 the sequence of reactivity is cationic approximately aromatic (stacking) > neutral > anionic; there is approximately an order of magnitude difference between the fastest reacting complex (k=10.2 x 10(-2) M(-1) s(-1)) and the slowest reacting complex (k=0.93 x 10(-2) M(-1) s(-1)) under these conditions. Platination of an oligodeoxynucleotide (30-mer), dsDNA, or an RNA transcript, shows that a Pt/nucleotide ratio between 1:10 and 1:20 (established by using flameless atomic absorption spectroscopy) results in probes with excellent hybridization characteristics. In terms of applicability and detection limits these platinated nucleic acid probes perform equally well compared to conventionally generated nucleic acid probes, that is, through enzymatic incorporation of covalently labeled nucleotide triphosphates. Applications of these reagents to in situ hybridization assays and gene expression profiling on microarrays illustrate the potential of these monofunctional binding platinum triamine compounds.

Animals↗

PubMed2001

Universal Linkage System: versatile nucleic acid labeling technique.

Over the last two decades nonradioactive nucleic acid labeling and detection systems have overcome the safety, disposal, stability and cost problems that are associated with radioactive techniques. Besides traditional, enzyme-mediated, nonradioactive labeling methods (e.g., random priming, nick translation or labeling by PCR), several chemical labeling systems have been developed (e.g., ULS, psoralen, alkylating agents). These methods provide fluorescent (or hapten) labeled probes for fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH) and microarray-based techniques. In this review the DNA-based molecular diagnostic applications and perspectives of the Universal Linkage System (ULS) technology will be described.

Chlamydia trachomatis↗

PubMed2000

New insights in the cellular processing of platinum antitumor compounds, using fluorophore-labeled platinum complexes and digital fluorescence microscopy.

The cellular distribution and processing pathways of two platinum compounds, modeling the antitumor drug cisplatin (cDDP) in human osteosarcoma (U2-OS) cells is reported. A [Pt(en)Cl] entity has been covalently linked to a carboxyfluorescein diacetate (CFDA) moiety and to a dinitrophenyl (DNP) moiety. The two different constructs were administered to living cell cultures that were analyzed using digital fluorescence microscopy. The non-fluorescent CFDA construct becomes fluorescent after cellular uptake and subsequent acetate hydrolysis by esterases, and is therefore suitable to monitor platinum in living cells; the DNP construct can be visualized by immunocytochemistry and consequently serves as a control. Both complexes were readily internalized by the cells, and localized throughout the whole cell. After 2-3 h the complex accumulated in the nucleus, but 6-8 h after incubation a punctuate staining of a cytoplasmic region was observed, that persisted and became more pronounced after 24 h. The overall fluorescence in the cell decreased over time, implying a secretion of the platinum complex. Surprisingly, the accumulation remained visible after 72 h. Co-localization experiments with a Golgi apparatus-selective stain indicate the involvement of Golgi vesicles in intracellular processing of cisplatin-derived complexes. Immunocytochemical studies, using the DNP derivative, resulted in very similar images as obtained with the CFDA construct. CFDA-boc (a non-platinum-containing fluorescein derivative) was used as control: a faint staining throughout the whole cell was observed. Cisplatin-resistant U2-OS/Pt cells showed staining patterns very similar to the U2-OS cells using both platinum constructs. This study illustrates that only a very small portion of the platinum complex eventually remains bound to DNA, as after 24 h no significant fluorescence could be observed in the nucleus. Cisplatin-derived complexes with fluorescent tags afford a new insight into the cellular processing of these complexes and therefore may contribute to further unraveling of the mechanism of platinum antitumor complexes.

Antineoplastic Agents↗

PubMed1999

The new anticancer drug [(2R)-aminomethylpyrrolidine](1, 1-cyclobutanedicarboxylato)platinum(II) and its toxic S enantiomer do interact differently with nucleic acids.

The interaction of the two chiral isomers of the new anticancer agent [Pt(ampyr)(cbdca)] (ampyr=aminomethylpyrrolidine, cbdca=cyclobutanedicarboxylate) with 5'-GMP and with short G-containing oligonucleotides has been studied using (1)H and (31)P NMR, UV-vis spectroscopy and molecular modelling. Each isomer loses the cbdca ligand upon binding to the DNA fragments. Two geometrical isomers of the DNA adducts are formed owing to the presence of the unsymmetric ampyr ligand. These isomers prove to be GG-N7,N7 chelates for d(GpG), d(pGpG) and d(CpGpG). A slight preference for the formation of one geometrical isomer is found in the case of DNA fragments having a phosphate moiety and/or a C base at the 5'-site of the GG sequence. H-bonding interactions from the NH(2) moiety towards the 5'-phosphate group and/or the O atom of the C base clearly favour the formation of one geometrical isomer. The presence of these H-bonds, together with the bulky pyrrolidine ring, has resulted in the unique observation (by (1)H NMR) of NH protons of coordinated amines that do not rapidly exchange in a 99.95% D(2)O solution. Temperature-dependence studies show an extremely slow stack <--> destack conformational change for the CGG adducts of the S isomer, which could be related to these stable H-bonds of the amine protons towards the oligonucleotide. For the R isomer this stack <--> destack conformational change is faster, probably owing to more steric hindrance of the pyrrolidine ring as deduced from the NOESY data, and as also suggested by molecular modelling. The observation of extremely slow rotation around the Pt-N7 bond for [Pt(R-ampyr)(GMP-N7)(2)] provides further evidence for increased steric hindrance of the R isomer compared to the S isomer. The rate of binding of the drug to G bases proved to be second order for both isomers; in fact the (toxic) S isomer is about two times more reactive than the (non-toxic) R isomer, as seen from k(2) values of 0.17+/-0.01 M(-1)s(-1)for [Pt(S-ampyr)(cbdca)] and 0.09+/-0.01 M(-1)s(-1) for [Pt(R-ampyr)(cbdca)]. No solvent-assisted pathway is involved in these reactions, since the complexes prove to be stable in solution for weeks and therefore only a direct attack of the G base on the Pt must be involved. Because hardly any intermediate species can be detected during the reaction, coordination of the second G base must occur much faster than the binding of the first G base. Since direct attack of the nucleobases takes place, steric interactions become extremely important and therefore are likely to determine the reactivity, activity and even the toxicity of such Pt complexes.

Antineoplastic Agents↗

PubMed1999

New strategy for multi-colour fluorescence in situ hybridisation: COBRA: COmbined Binary RAtio labelling.

Multicolour in situ hybridisation (MFISH) is increasingly applied to karyotyping and detection of chromosomal abnormalities. So far 27 colour analyses have been described using fluorescently labelled chromosome painting probes in a so-called combinatorial approach. In this paper a new strategy is presented to use efficiently the currently available number of spectrally separated fluorophores in order to increase the multiplicity of MFISH. We introduce the principle of COBRA (COmbined Binary RAtio labelling), which is based on the simultaneous use of combinatorial labelling and ratio labelling. Human chromosome painting in 24 colours is accomplished using four fluorophores only. Three fluorophores are used pair wise for ratio labelling of a set of 12 chromosome painting probes. The second set of 12 probes is labelled identically but is also given a binary label (fourth fluorophore). The COBRA method is demonstrated on normal human chromosomes and on a lymphoma (JVM) cell line, using probes enzymatically labelled with fluorescein, lissamine and cy5 as primary fluorophores, and diethylaminocoumarin (DEAC), a blue dye, as combinatorial fourth label to demonstrate incorporated digoxigenin. In addition, the principle was tested using chemical labelling. The first set of 12 painting probes was therefore labelled by ULS (Universal Linkage System), using DEAC, cy3 and cy5 as primary labels, and the second set was labelled similarly, but also contained a digoxigenin-ULS label, which was indirectly stained with fluorescein. Subsequently, a mathematical analysis is presented and methods are indicated for achieving an MFISH multiplicity of 48, 96 or even higher using existing technology.

Chromosomes, Human↗

PubMed1999

ULS: a versatile method of labeling nucleic acids for FISH based on a monofunctional reaction of cisplatin derivatives with guanine moieties.

The broad extension of an existing chemical DNA labeling technique for molecular cytogenetics is described. Called the Universal Linkage System (ULS(TM)), it is based on the capability of monoreactive cisplatin derivatives to react at the N7 position of guanine moieties in DNA. Simple repetitive probes, cosmids, PACs, and chromosome-specific painting probes were labeled by ULS and used in a series of multicolor fluorescence in situ hybridization experiments on interphase and metaphase cells. It is demonstrated that ULS-labeled probes, in general, perform as well as the more conventional enzymatically labeled probes. The advantage of ULS labeling over enzymatic labeling techniques is that it is a fast and simple procedure, and that the labeling can easily be scaled up for bulk probe synthesis. In addition, with ULS labeling it is possible to label degraded DNA, a situation in which enzymatic labeling is known to perform unsatisfactorily.

B-Lymphocytes↗