Histone deacetylase inhibitor sodium butyrate enhances cellular radiosensitivity by inhibiting both DNA nonhomologous end joining and homologous recombination.

HDAC inhibitors have been proposed as radiosensitizers in cancer therapy. Their application would permit the use of lower radiation doses and would reduce the adverse effects of the treatment. However, the molecular mechanisms of their action remain unclear.

Sodium butyrate enhances the cytotoxic effect of cisplatin by abrogating the cisplatin imposed cell cycle arrest.

Background: Histone deacetylase inhibitors have been proposed as potential enhancers of the cytotoxic effect of cisplatin and other anticancer drugs. Their application would permit the use of lower therapeutic doses and reduction of the adverse side effects of the drugs. However, the molecular mechanisms by which they sensitize the cells towards anticancer drugs are not known in details, which is an obstacle in developing effective therapeutic protocols.

Tumor-specific protein human galectin-1 interacts with anticancer agents.

The present work shows a novel binding activity of the tumor specific lectin--recombinant human galectin-1 (hGal-1)--to three porphyrin compounds: (1) Zn-porphyrin (ZnTPPS); (2) Mn-porphyrin and (3) Au-porphyrin. These compounds are widely applied in the photodynamic therapy of cancer (PDT). Our data indicate that hGal-1, similar to some plant lectins, a bacterial lectin from Pseudomonas aeruginosa and an animal lectin from Helix pomatia, possesses dual functions binding to both carbohydrate and non-carbohydrate ligands.

Comparison of the global genomic and transcription-coupled repair rates of different lesions in human cells.

There are two subclasses of nucleotide excision repair (NER). One is the global genomic repair (GGR) which removes lesions throughout the genome regardless of whether any specific sequence is transcribed or not. The other is the transcription-coupled repair (TCR), which removes lesions only from the transcribed DNA sequences.

DNA interstrand crosslinks repair in mammalian cells.

We studied the formation of double strand breaks (DSBs) as intermediates in the repair of DNA interstrand crosslinks (ICLs) by homologous recombination (HR). The plasmid EGFP-N1 was crosslinked with trioxsalen to give one ICL per plasmid on average. HeLa cells were transfected with the crosslinked plasmids and the ICL repair was monitored by following the restoration of the GFP expression.

Fluorescence study of steroid hormone binding activity of Helix pomatia agglutinin.

Helix pomatia agglutinin (HPA) is a N-acetylgalactosamine (GalNAc) binding lectin, found in the reproductive gland of a Roman snail. The present study has shown that HPA, in addition to its carbohydrate binding capacity possesses a hydrophobic binding activity. This protein binds with high affinity (k(D)=1.

Enhanced repair of DNA interstrand crosslinks in S phase.

Hela cells synchronized in G1 and S phases of the cell cycle were transfected with pEGFP crosslinked with trioxsalen. Twelve hours later the number of fluorescent cells was determined by fluorescent microscopy. Cells in S phase have repaired 0.

Nuclear matrix support of DNA replication.

In higher eukaryotic cells, DNA is tandemly arranged into 10(4) replicons that are replicated once per cell cycle during the S phase. To achieve this, DNA is organized into loops attached to the nuclear matrix. Each loop represents one individual replicon with the origin of replication localized within the loop and the ends of the replicon attached to the nuclear matrix at the bases of the loop.

Repair of DNA interstrand crosslinks may take place at the nuclear matrix.

Host cell reactivation assay using Trioxsalen-crosslinked plasmid pEGFP-N1 showed that human cells were able to repair Trioxsalen interstrand crosslinks (ICL). To study the mechanism of this repair pathway, cells were transfected with the plasmids pEGFP-1, which did not contain the promoter of the egfp gene, and with pEGFP-G-, which did not contain the egfp gene. Neither of these plasmids alone was able to express the green fluorescent protein.

Nucleotide excision repair rates in rat tissues.

We have determined and compared nucleotide excision repair capability of several rat tissues by a method based on restoration of the transformation activity of UV-irradiated pBlueScript by incubation in repair-competent protein extracts. After 3 h of incubation, plasmid DNA was isolated and used to transform competent Escherichia coli cells. Damaged plasmids showed low transformation efficiency prior to incubation in repair-competent extracts.

Relationship between DNA repair capacity and resistance to genotoxins in four human cell lines.

We have developed fast, reliable and simple fluorescent method to assess and compare repair capacity of cells. To this end plasmid pEGFP containing the gene for the enhanced green fluorescent protein was damaged in vitro by genotoxic agents and introduced into cells by transfection. The repair capacity of the cells was determined from the number of fluorescent cells counted with a fluorescent microscope 24 h after transfection.

DNase I sensitive site in the core region of the human beta-globin origin of replication.

HeLa cells were synchronized at late G1, early S, and late S phase of the cell cycle by nocodazole treatment. The cells were permeabilized with Triton X-100, digested with DNAse I, and extracted with 0.2 M ammonium sulfate to remove the digested chromatin.

Dynamics of association of origins of DNA replication with the nuclear matrix during the cell cycle.

DNA of replication foci attached to the nuclear matrix was isolated from Chinese hamster ovary cells and human HeLa cells synchronized at different stages of the G(1) and S phases of the cell cycle. The abundance of sequences from dihydrofolate reductase ori-beta and the beta-globin replicator was determined in matrix-attached DNA. The results show that matrix-attached DNA isolated from cells in late G(1) phase was enriched in origin sequences in comparison with matrix-attached DNA from early G(1) phase cells.

Chronic renal failure and anterior hypophysial hormones.

Kidneys are not only organs with an excretory function but they produce their own endocrine factors which are involved in supporting homeostasis in the organism. The kidneys are the organs in which metabolism and biodegradation of many hormones take place. Together with the liver, the kidneys actively take part in the catabolism of hormones.

Distribution of DNA replication origins between matrix-attached and loop DNA in mammalian cells.

Using a previously developed procedure (Gencheva et al. [1996] J Biol Chem 271:2608-2614), we isolated a DNA fraction consisting of short fragments originating from the regions of initiation of DNA synthesis from exponentially growing Chinese hamster ovary cells. This fraction arbitrarily designated as "collective origin fraction" was labeled in vitro and used to probe the abundance of origin containing sequences in preparations of matrix-attached and loop DNA isolated by two different procedures from Chinese hamster ovary cells.

Two-wavelength fluorescence assay for DNA repair.

A simple and reliable quantitative assay for measuring cellular DNA repair capacity has been developed. It is based on the host cell reactivation of the UV-irradiated plasmid pEGFP carrying the marker gene for the enhanced green fluorescent protein (EGFP). As a reference we used the plasmid pEYFP carrying the gene for a red-shifted fluorescent protein (EYFP).

Iron(II)-mimosine catalyzed cleavage of DNA.

Mimosine, DNA breaKs, Free Radicals, Fenton Reaction Supercoiled plasmid DNA was treated in vitro with H2O2, DTT and either Fe (II), Fe (II)-EDTA or Fe (II)-mimosine. The rate of DNA break formation was followed by the conversion of the supercoiled form into relaxed-circular and linear forms. In the concentration interval of 0-4 microM Fe (II), Fe (II)-EDTA slowed-down the formation of DNA breaks, while Fe (II)-mimosine enhanced the rate of break formation up to several times.

Influence of dopamine energic pharmacology drugs on secretion of the adrenocorticotropic hormone from the hypophysis.

To elucidate the role of dopamine as a neuromediator in the adrenocorticotropic hormone (ACTH) secretion, investigations were carried out with dopaminergic pharmacology drugs on male white Wistar rats. In the first series of experiments, the effects of 200 mg/kg body wt L-DOPA, of the combination of 200 mg/kg L-DOPA and 50 mg/kg body wt carbidopa, and of 2.5 mg/kg body wt bromocriptine, after a single intraperitoneal injection of ACTH in the serum of rats after 30, 90 and 120 min, following the injection, were studied.

Regulation of DNA synthesis by the higher-order chromatin structure.

Mouse erythroleukemia cells were treated with the topoisomerase II poison VP-16, the intrastrand crosslinking agent cis-DDP, and the ribonucleotide reductase inhibitor hydroxyurea. In all cases, the rate of DNA synthesis decreased as a result of the treatment. To study the mechanism of inhibition of DNA chain elongation, we determined DNA synthesis in a cell-free replication system containing isolated nuclei and cytoplasmic extracts.

Treatment of mammalian cells with mimosine generates DNA breaks.

Exponentially growing mouse erythroleukemia (MEL) cells and quiescent human peripheral blood lymphocytes (PBL) were treated with different concentrations of the nonprotein amino acid mimosine for 16 h. The treatment of the cycling cell population with 400 microM mimosine caused inhibition of DNA replication, changes in the progression of the cells in the cell cycle, and apoptosis. Nucleoid sedimentation analysis and comet assay were used to monitor the appearance and accumulation of DNA breaks.

Transcriptionally active and inactive mouse beta-globin gene loci are repaired at similar rates after ultraviolet irradiation.

It has been demonstrated, by Northern blot and in situ hybridization, that the mouse erythroleukaemia cell line F4N-Sofia constitutively expresses the beta-globin genes. The recently developed quantitative assay for DNA repair has been used to study the overall repair rate in the beta-globin gene domain in this cell line after ultraviolet irradiation and to compare it with the repair rate of the same chromatin domain in mouse Ehrlich ascites tumour cells which do not express the beta-globin genes. The results showed that in both cases the 5'-end of the domain was repaired preferentially and that the repair rates in the two cell lines were very similar despite the different transcription state of the genes.

Comparison of repair activity in different genomic regions.

We have developed a quantitative technique to determine repair activity at defined genomic regions. Cells were treated with hydroxyurea to inhibit the replicative DNA synthesis and were incubated with 5-bromodeoxyuridine (BrdUrd) to label the regions undergoing repair. In the course of the labelling, the regions that were more actively repaired would incorporate more BrdUrd than the regions that were less actively repaired.