Emerin increase in regenerating muscle fibers.

The fate of emerin during skeletal muscle regeneration was investigated in an animal model by means of crush injury. Immunofluorescence, immunoblotting and mRNA analysis demonstrated that emerin level is increased in regenerating rat muscle fibers with respect to normal mature myofibers. This finding suggests an involvement of emerin during the muscle fiber regeneration process, in analogy with its reported involvement in muscle cell differentiation in vitro. The impairment of skeletal muscle physiological regeneration or reorganization could be a possible pathogenetic mechanism for Emery Dreifuss muscular dystrophy.

Immunocytochemistry of nuclear domains and Emery-Dreifuss muscular dystrophy pathophysiology.

The present review summarizes recent cytochemical findings on the functional organization of the nuclear domains, with a particular emphasis on the relation between nuclear envelope-associated proteins and chromatin. Mutations in two nuclear envelope-associated proteins, emerin and lamin A/C cause the Emery-Dreifuss muscular dystrophy; the cellular pathology associated with the disease and the functional role of emerin and lamin A/C in muscle cells are not well established. On the other hand, a large body of evidence indicates that nuclear envelope-associated proteins are involved in tissue-specific gene regulation. Moreover, chromatin remodeling complexes trigger gene expression by utilizing the nuclear matrix-associated actin, which is known to interact with both emerin and lamin A/C. It is thus conceivable that altered expression of these nuclear envelope-associated proteins can account for an impairment of gene expression mainly during cell differentiation as suggested by recent experimental findings on the involvement of emerin in myogenesis. The possibility that Emery-Deifuss muscular dystrophy pathogenesis could involve alteration of the signaling pathway is considered.

Staurosporine treatment and serum starvation promote the cleavage of emerin in cultured mouse myoblasts: involvement of a caspase-dependent mechanism.

Emerin is a nuclear membrane-anchored protein which is absent or mutated in patients affected by Emery-Dreifuss muscular dystrophy. In this study, we induced apoptosis in cultured mouse myoblasts to evaluate emerin fate during the nuclear destabilization involved in programmed cell death. Emerin proteolysis was observed in myocytes during the apoptotic process. Myoblast apoptosis and emerin degradation were associated with chromatin compaction and detachment from the nuclear lamina, as detected by electron microscopy. In vivo specific inhibition of caspase 3 or caspase 6 activity completely abolished emerin proteolysis. These results show that the process of programmed cell death in muscle cells leads to emerin proteolysis, which appears to be related to caspase 6 activation and to cleavage of other nuclear envelope proteins, that share sequence homologies or functional features with emerin.

[2,1-c][1,4]benzodiazepine (PBD)-distamycin hybrid inhibits DNA binding to transcription factor Sp1.

We designed and synthesized the hybrid 6, prepared combining the minor groove binders distamycin A and pyrrolo [2,1-c][1,4] benzodiazepine (PBD) 4, related to the natural occurring anthramycin (2) and DC-81 (3). In this paper, the effects of the compound 6 on molecular interactions between DNA and transcription factor Sp1 were studied. The results obtained demonstrate that PBD-distamycin hybrid is a powerful inhibitor of Sp1/DNA interactions.

Pyrazolo-triazoles as light activable DNA cleaving agents.

In view of the continuous interest in new DNA cleaving compounds, both for the development of new therapeutic agents and for the possible use as reagents in nucleic acids research, a few pyrazolo[3,4-d][1,2,3]triazole derivatives have been obtained and investigated for their antiproliferative activity and capability to cleave DNA, after light-activation. A possible in situ activation, i.e. in neoplastic tissues, of less cytotoxic derivatives, may lead to potential antitumor compounds endowed with high therapeutic indexes.

Biospecific interaction analysis (BIA) of low-molecular weight DNA-binding drugs.

DNA-binding drugs have been reported to be able to interfere with the activity of transcription factors in a sequence-dependent manner, leading to alteration of transcription. This and similar effects could have important practical applications in the experimental therapy of many human pathologies, including neoplastic diseases and viral infections. The analysis of the biological activity of DNA-binding drugs by footprinting, gel retardation, polymerase chain reaction, and in vitro transcription studies does not allow a real time study of binding to DNA and dissociation of the generated drugs/DNA complexes. The recent development of biosensor technologies for biospecific interaction analysis (BIA) enables monitoring of a variety of molecular reactions in real-time by surface plasmon resonance (SPR). In this study, we demonstrate that molecular interactions between DNA-binding drugs (chromomycin, mithramycin, distamycin, and MEN 10567) and biotinylated target DNA probes immobilized on sensor chips is detectable by SPR technology using a commercially available biosensor. The target DNA sequences were synthetic oligonucleotides mimicking the Sp1, NF-kB, and TFIID binding sites of the long terminal repeat of the human immunodeficiency type 1 virus. The results obtained demonstrate that mithramycin/DNA complexes are less stable than chromomycin/DNA complexes; distamycin binds to both NF-kB and TATA box oligonucleotides, but distamycin/(NF-kB)DNA complexes are not stable; the distamycin analog MEN 10567 binds to the NF-kB mer and the generated drug/DNA complexes are stable. The experimental approach described in this study allows fast analysis of molecular interactions between DNA-binding drugs and selected target DNA sequences. Therefore, this method could be used to identify new drugs exhibiting differential binding activities to selected regions of viral and eukaryotic gene promoters.

Synthesis, in vitro antiproliferative activity, and DNA-binding properties of hybrid molecules containing pyrrolo[2,1-c][1, 4]benzodiazepine and minor-groove-binding oligopyrrole carriers.

The synthesis, biological activity, and DNA-binding properties of a series of four hybrids prepared by combining polypyrrole minor groove binders and pyrrolo[2,1-c][1,4]benzodiazepine (PBD) 13, related to the naturally occurring anthramycin (3) and DC-81 (4), have been described, and structure-activity relationships have been discussed. These hybrids 22-25 contain from one to four pyrrole units, respectively. To investigate sequence selectivity and stability of drug/DNA complexes, DNase I footprinting and arrested polymerase chain reaction (PCR) were performed on human c-myc oncogene, estrogen receptor gene, and human immunodeficiency virus type 1 long terminal repeat (HIV-1 LTR) gene sequences. The antiproliferative activity of the hybrids has been tested in vitro on human myeloid leukemia K562 and T-lymphoid Jurkat cell lines and compared to antiproliferative effects of the natural product distamycin A 1, its tetrapyrrole homologue 17, DC 81 (4), and the PBD methyl ester 12. The results obtained demonstrate that the hybrids 22-25 exhibit different DNA-binding activity with respect to both distamycin A 1 and PBD 12. In addition, a direct relationship was found between number of pyrrole rings present in the hybrids 22-25 and stability of drug/DNA complexes. With respect to antiproliferative effects, it was found that the increase in the length of the polypyrrole backbone leads to an increase of in vitro antiproliferative effects, i.e., the hybrid 25 containing the four pyrroles is more active than 22, 23, and 24 both against K562 and Jurkat cell lines.

Interaction of the human NF-kappaB p52 transcription factor with DNA-PNA hybrids mimicking the NF-kappaB binding sites of the human immunodeficiency virus type 1 promoter.

We determined whether peptide nucleic acids (PNAs) are able to interact with NF-kappaB p52 transcription factor. The binding of NF-kappaB p52 to DNA-DNA, DNA-PNA, PNA-DNA, and PNA-PNA hybrid molecules carrying the NF-kappaB binding sites of human immunodeficiency type 1 long terminal repeat was studied by (i) biospecific interaction analysis (BIA) using surface plasmon resonance technology, (ii) electrophoretic mobility shift, (iii) DNase I footprinting, and (iv) UV cross-linking assays. Our results demonstrate that NF-kappaB p52 does not efficiently bind to PNA-PNA hybrids. However, a DNA-PNA hybrid molecule was found to be recognized by NF-kappaB p52, although the molecular complexes generated exhibited low stability. From the theoretical point of view, our results suggest that binding of NF-kappaB p52 protein to target DNA motifs is mainly due to contacts with bases; interactions with the DNA backbone are, however, important for stabilization of the protein-DNA complex. From the practical point of view, our results suggest that DNA-PNA hybrid can be recognized by NF-kappaB p52 protein, although with an efficiency lower than DNA-DNA NF-kappaB target molecules; therefore, our results should encourage studies on modified PNAs in order to develop potential agents for the decoy approach in gene therapy.

Analysis of the human HLA-DRA gene upstream region: evidence for a stem-loop array directed by nuclear factors.

Sequence analysis of the far-upstream region of the human HLA-DRA gene has revealed the presence of Y' and X' boxes, highly homologous to the well characterized Y and X boxes present within the proximal-promoter region. Comparison of Y, Y', X, and X' box sequences present within different class II MHC genes of different species demonstrates that these boxes are conserved during evolution, suggesting an important role in regulation of gene expression. The far-upstream region and the proximal promoter region of the class II MHC genes could be organized in secondary structures, as suggested for the EA gene, the murine counterpart of the human HLA-DRA gene. The essential feature of this model is a dimerization of the proteins binding to X and X' and/or Y and Y' boxes resulting in a loop-out of the intervening DNA and a rapprochement of the far-upstream and proximal-promoter regions, and consequently of any proteins binding to them. We set up an in vitro approach in order to determine whether proteins bound to sequences present within far-upstream and proximal-promoter regions of the human HLA-DRA gene could direct a secondary structure assembly of regulative regions. Moreover, by gel retardation and DNase I footprinting assays, we demonstrate that similar proteins bind to Y and Y' boxes and, among these proteins, NF-Y was unambiguously identified by antibody-super shift experiments. Taken together, the data presented in this paper provide evidence supporting the hypothesis that a stem-loop array of the 5'-upstream region of the human HLA-DRA gene could be directed by nuclear factors. In this manner, additional nuclear factors bound to the far region could be driven in close proximity of the transcription initiation site.

PNU 157977: a new potent antitumour agent exhibiting low in vivo toxicity in mice injected with L1210 leukaemia cells.

The design, synthesis, in vitro and in vivo activity against L1210 murine leukaemia of the dibromo nitrogen mustard derivative of 2, called PNU 157977, is described and the structure-activity relationship discussed. This dibromo derivative is almost two orders of magnitude more cytotoxic than the dichloro counterpart having the same oligopeptidic chain (IC50 2.7 ng/ml versus 225 ng/ml), and it showed in vivo an increased survival time which is 5- and 3-fold longer than that of tallimustine and 2 (and T/C 750 versus 133 and 213) respectively. Moreover PNU 157977 shows activity against the M5076 solid tumour markedly inferior to that of the closely analogous 2. Footprinting experiments conducted using the oestrogen receptor PCR probe as the footprinting target molecule show that PNU 157977 possesses a different sequence-specific alkylation and greater cleavage activity than either 2 or tallimustine.

The DNA-binding drugs mithramycin and chromomycin are powerful inducers of erythroid differentiation of human K562 cells.

The human leukaemic K562 cell line can be induced in vitro to undergo erythroid differentiation by a variety of chemical compounds, including haemin, butyric acid, 5-azacytidine and cytosine arabinoside. Differentiation of K562 cells is associated with an increased expression of embryo-fetal globin genes, such as the zeta, epsilon and gamma globin genes. Therefore the K562 cell line has been proposed as a useful in vitro model system to determine the therapeutic potential of new differentiating compounds as well as to study the molecular mechanism(s) regulating changes in the expression of embryonic and fetal human globin genes. Inducers of erythroid differentiation which stimulate gamma-globin synthesis could be considered for possible use in the experimental therapy of those haematological diseases associated with a failure in the expression of adult beta-globin genes. In this paper we demonstrated that the G + C selective DNA-binding drugs chromomycin and mithramycin were powerful inducers of erythroid differentiation of K562 cells. Erythroid differentiation was associated with an increase in the accumulation of (a) Hb Gower 1 and Hb Portland and (b) gamma-globin mRNA.

Surface plasmon resonance for real-time monitoring of molecular interactions between a triple helix forming oligonucleotide and the Sp1 binding sites of human Ha-ras promoter: effects of the DNA-binding drug chromomycin.

DNA-binding molecules have been recently proposed as potential inhibitors of molecular interactions between transcription factors and target DNA sequences. Among DNA-binding drugs, chromomycin binds to GC-rich sequences of the Sp1 binding sites of the Ha-ras oncogene. These sites are also molecular targets of a triple-helix forming oligonucleotide [Sp1(Ha-ras)TFO] which is able to inhibit Ha-ras oncogene transcription. We studied molecular interactions between triple-helix forming oligonucleotides and target Sp1 binding sites of the human Ha-ras promoter in the presence of the DNA-binding drug chromomycin. This study was performed by (a) surface plasmon resonance and biosensor technology, (b) gel retardation assay and (c) magnetic capturing of molecular complexes between TFO, chromomycin and target DNA. The main conclusion of our study is that low concentrations of chromomycin allow binding of the triplex-forming oligonucleotide to Sp1 target DNA sequences of the Ha-ras oncogene promoter. Higher concentrations of this DNA-binding drug fully suppress molecular interactions between the Sp1(Ha-ras)TFO and target DNA. Additionally, low concentrations of chromomycin potentiate the effects of the Sp1(Ha-ras)TFO in inhibiting the molecular interactions between purified Sp1 transcription factor and target DNA sequences.

Biosensor technology and surface plasmon resonance for real-time detection of HIV-1 genomic sequences amplified by polymerase chain reaction.

BACKGROUND: The recent development of biosensor technologies for biospecific interaction analysis enables the monitoring of a variety of molecular reactions in real time by surface plasmon resonance (SPR). If the ligand is a biotinylated single stranded DNA, this technology could monitor DNA-DNA hybridization. This approach could be of great interest in virology, since the hybridization step is oftenly required to confirm specificity of molecular diagnosis. OBJECTIVES: To determine whether real-time molecular diagnosis of human immunodeficiency virus type I (HIV-1) could be performed using biosensors and SPR technology. STUDY DESIGN: Specific hybridization of a biotinylated HIV-1 oligonucleotide probe immobilized on a sensor chip to single stranded DNA obtained by asymmetric polymerase-chain reaction (PCR) was determined using the BIAcore biosensor. RESULTS: Direct injection of asymmetric PCR to a sensor chip carrying an internal HIV-1 oligonucleotide probe allows detection of hybridization by SPR using biosensor technology. This enabled us to apply a real-time, one-step, non-radioactive protocol to demonstrate the specificity of amplification of HIV-1 genomic sequences by PCR. CONCLUSION: The procedure described in this study for HIV-1 detection is simple, fast (PCR and SPR analyses take 30 min), reproducible and could be proposed as an integral part of automated diagnostic systems based on the use of laboratory workstations and biosensors for DNA isolation, preparation of PCR reactions and analysis of PCR products.

Targeting of the HIV-1 long terminal repeat with chromomycin potentiates the inhibitory effects of a triplex-forming oligonucleotide on Sp1-DNA interactions and in vitro transcription.

We have studied the effects of chromomycin and of a triple-helix-forming oligonucleotide (TFO) that recognizes Sp1 binding sites on protein-DNA interactions and HIV-1 transcription. Molecular interactions between chromomycin, the Sp1 TFO and target DNA sequences were studied by gel retardation, triplex affinity capture using streptavidin-coated magnetic beads and biosensor technology. We also determined whether chromomycin and a TFO recognizing the Sp1 binding sites of the HIV-1 long terminal repeat (LTR) inhibit the activity of restriction enzyme HaeIII, which recognizes a sequence (5'-GGCC-3') located within these Sp1 binding sites. The effects of chromomycin and the TFO on the interaction between nuclear proteins or purified Sp1 and a double-stranded oligonucleotide containing the Sp1 binding sites of the HIV-1 LTR were studied by gel retardation. The effects of both chromomycin and TFO on transcription were studied by using an HIV-1 LTR-directed in vitro transcription system. Our results indicate that low concentrations of chromomycin potentiate the effects of the Sp1 TFO in inhibiting protein-DNA interactions and HIV-1-LTR-directed transcription. In addition, low concentrations of chromomycin do not affect binding of the TFO to target DNA molecules. The results presented here support the hypothesis that both DNA binding drugs and TFOs can be considered as sequence-selective modifiers of DNA-protein interactions, possibly leading to specific alterations of biological functions. In particular, the combined use of chromomycin and TFOs recognizing Sp1 binding sites could be employed in order to abolish the biological functions of promoters (such as the HIV-1 LTR) whose activity is potentiated by interactions with the promoter-specific transcription factor Sp1.

In vitro and in vivo binding of a CC-1065 analogue to human gene sequences: a polymerase-chain reaction study.

In this paper we analyse the in vitro sequence selectivity of the CC-1065 analogue 2-[[5-[(1H-indol-2-yl]carbonyl)-1H-indol-2-yl] carbonyl]-7-methyl-1,2,8,8a-tetrahydrocyclopropa [c]-pyrrolo-[3,2-e]-indol-4-one (U-71184) employing the polymerase-chain reaction (PCR). In addition, we determined whether alteration of PCR by U-71184 is detected when DNA is isolated from cells cultured in the presence of this drug. As molecular model systems we employed the human estrogen receptor gene, the Ha-ras oncogene and the chromosome X-linked, (CGG)-rich fragile X mental retardation-1 gene. The first conclusion that can be drawn from the experiments reported in our paper is that U-71184 inhibits PCR in a sequence-dependent manner. A second conclusion of our experiments is that PCR performed on DNA from U-71184-treated cells is inhibited when the primers amplifying the estrogen receptor gene region are used. This approach might bring important information on both in vivo uptake of the drug by target cells and binding to DNA.

Surface plasmon resonance for real-time detection of molecular interactions between chromomycin and target DNA sequences.

DNA-binding drugs interfere with the activity of a large variety of transcription factors, leading to an alteration of transcription. This and similar effects could have important practical applications in the experimental therapy of many human pathologies, including neoplastic diseases. The analysis of sequence selectivity of DNA-binding drugs by footprinting, gel retardation studies, polymerase chain reaction and in vitro transcription does not allow an easy study of kinetics of binding and dissociation. The recent development of biosensor technologies for biospecific interaction analysis (BIA) enables the monitoring of a variety of molecular reactions in real-time by surface plasmon resonance (SPR). In this report we demonstrate that molecular interactions between the DNA-binding drug chromomycin and a biotinylated GC-rich Ha-ras oligonucleotide probe immobilized on a sensor chip is detectable by SPR technology using the BIAcore(TM) biosensor. This approach appears of interest in the development of drugs exhibiting differential affinity for target DNA sequences for the following reasons: a) results are obtained within one hour; b) unlike footprinting and gel retardation studies, this technology does not require P-32-labelled probes; c) BIA allows kinetic studies of both association and dissociation.

Targeting of the Sp1 binding sites of HIV-1 long terminal repeat with chromomycin. Disruption of nuclear factor.DNA complexes and inhibition of in vitro transcription.

Sequence selectivity of DNA-binding drugs has recently been reported in a number of studies employing footprinting and gel retardation approaches. In this paper, we studied the biochemical effects of the sequence-selective binding of chromomycin to the long terminal repeat of the human immunodeficiency type I virus. Deoxyribonuclease I (E.C.3.1.21.1) footprinting, arrested polymerase chain reaction, gel retardation and in vitro transcription experiments have demonstrated that chromomycin preferentially interacts with the binding sites of the promoter-specific transcription factor Sp1. Accordingly, interactions between nuclear proteins and Sp1 binding sites are inhibited by chromomycin, and this effect leads to a sharp inhibition of in vitro transcription.

Sequencing of an upstream region of the human HLA-DRA gene containing X' and Y' boxes.

In this paper we report the characterization of a newly sequenced 5' upstream region of the human HLA-DRA gene. We performed (i) search for transcription factor motifs, (ii) analysis of CpG display and observed/expected frequency ratios, (iii) search for regions homologous to the 5' upstream sequences of the murine EA gene, (iv) DNase I footprinting experiments and (v) electrophoretic mobility shift assays. Our results demonstrate the existence, in the HLA-DRA gene, of Y' and X' boxes highly homologous to the Y and X boxes present in MHC class II genes, but oriented in the opposite direction. These Y' and X' boxes have been conserved during the molecular evolution of both human HLA-DRA and murine EA genes. DNase I footprinting and gel retardation experiments suggest that the X' and Y' boxes of the HLA-DRA upstream gene region are specifically recognized by nuclear proteins that also bind to the X and Y boxes of the HLA-DRA proximal promoter, respectively.