Therapeutic potential of nucleic acid-based drugs in coronary hyper- proliferative vascular diseases.

The thickening of the vessel wall (intimal hyperplasia) is a pathological process which often follows revascularization approaches such as transluminal angioplasty and artery bypass graft, procedures used to re-vascularize stenotic artery. Despite the significant improvements in the treatment of intimal hyperplasia obtained in the last years, the problem has not completely solved. Nucleic acid based-drugs (NABDs) represent an emergent class of molecules with potential therapeutic value for the treatment of intimal hyperplasia. NABDs of interest in the field of intimal hyperplasia are: ribozymes, DNAzymes, antisense oligonucleotides, decoy oligonucleotides, small interfering RNAs and micro interfering RNAs. These molecules can recognize, in a sequencespecific fashion, a target which, depending on the different NABDs, can be represented by a nucleic acid or a protein. Upon binding, NABDs can down-modulate the functions of the target (mRNA/proteins) and thus they are used to impair the functions of disease-causing biological molecules.In spite of the great therapeutic potential demonstrated by NABDs in many experimental model of intima hyperplasia, their practical use is hindered by the necessity to identify optimal delivery systems to the vasculature. In the first part of this review a brief description of the clinical problem related to intima hyperplasia formation after revascularization procedures is reported. In the second part, the attention is focused on the experimental evidences of NABD therapeutic potential in the prevention of intimal hyperplasia. Finally, in the third part, we will describe the strategies developed to optimize NABD delivery to the diseased vessel.

Dissecting the expression of EEF1A1/2 genes in human prostate cancer cells: the potential of EEF1A2 as a hallmark for prostate transformation and progression.

BACKGROUND: In prostate adenocarcinoma, the dissection of the expression behaviour of the eukaryotic elongation factors (eEF1A1/2) has not yet fully elucidated. METHODS: The EEF1A1/A2 expressions were investigated by real-time PCR, western blotting (cytoplasmic and cytoskeletal/nuclear-enriched fractions) and immunofluorescence in the androgen-responsive LNCaP and the non-responsive DU-145 and PC-3 cells, displaying a low, moderate and high aggressive phenotype, respectively. Targeted experiments were also conducted in the androgen-responsive 22Rv1, a cell line marking the progression towards androgen-refractory tumour. The non-tumourigenic prostate PZHPV-7 cell line was the control. RESULTS: Compared with PZHPV-7, cancer cells showed no major variations in EEF1A1 mRNA; eEF1A1 protein increased only in cytoskeletal/nuclear fraction. On the contrary, a significant rise of EEF1A2 mRNA and protein were found, with the highest levels detected in LNCaP. Eukaryotic elongation factor 1A2 immunostaining confirmed the western blotting results. Pilot evaluation in archive prostate tissues showed the presence of EEF1A2 mRNA in near all neoplastic and perineoplastic but not in normal samples or in benign adenoma; in contrast, EEF1A1 mRNA was everywhere detectable. CONCLUSION: Eukaryotic elongation factor 1A2 switch-on, observed in cultured tumour prostate cells and in human prostate tumour samples, may represent a feature of prostate cancer; in contrast, a minor involvement is assigned to EEF1A1. These observations suggest to consider EEF1A2 as a marker for prostate cell transformation and/or possibly as a hallmark of cancer progression.

The expression levels of the translational factors eEF1A 1/2 correlate with cell growth but not apoptosis in hepatocellular carcinoma cell lines with different differentiation grade.

Despite the involvement of the elongation factors eEF1A (eEF1A1 and eEF1A2) in the development of different cancers no information is available on their possible contribution to the biology of hepatocellular carcinoma (HCC). We investigated the expression of both forms of eEF1A in HepG2 and JHH6 cell lines considered to be a good in vitro model of HCC at different stage of differentiation. Our data indicate that the mRNA amount of eEF1A1 is increased in both cell lines as compared to normal liver tissue, but eEF1A2 mRNA level is markedly increased only in JHH6. Moreover, the less differentiated cell line JHH6 displays higher EEF1A1 and EEF1A2 mRNAs levels and an higher nuclear-enriched/cytoplasm ratio of EEF1A protein compared to the better differentiated HepG2 cell line. Over-expression depends only partially on gene amplification. The more abundant mRNA levels and the higher nuclear-enriched/cytoplasm ratio of eEF1A in JHH6 neither correlate with apoptosis resistance nor with proliferation rate in sub-confluent cells. However, in confluent cells, a clear tendency to maintain JHH6 into the cell cycle was observed. In conclusion, we document the increased mRNA levels of EEF1A genes in HCC cell lines compared to normal liver. Additionally, we show the increased nuclear-enriched/cytoplasmic protein ratio of eEF1A and the marked raise of the expression of both eEF1A forms in JHH6 compared to HepG2, suggesting the possibility that eEF1A forms might become a relevant markers related to HCC tumor phenotype.

Anxiety stress and nociceptive responses in mice.

Nociception in laboratory animals appears to be influenced by physical or emotional stressors. Nevertheless, the reported data are not univocal. Discrepancies seem to be caused by some kind of stress model and/or by the timing of stressor application. The aim of the present work is to study the influence of chronic application of a well-controlled and defined anxiety stress paradigm (rotational stress) on the behavioral formalin pain responses in mice maintained in a low-stress environment. The results indicate that emotional chronic stress increases specific pain responses in the late inflammatory phase and, correspondingly, decreases self-grooming. Locomotor activity appears influenced by pain presence only. The hormonal and neural mechanisms that could be involved in the observed nonspecific and specific nociceptive responses to stress are discussed.

Alpha-anomeric configuration of GT oligodeoxynucleotide leads to loss of the specific aptameric and cytotoxic properties retained by the beta-anomeric analog.

The development of antisense, antigene, or aptameric oligonucleotides to modulate in vivo cellular functions depends on using stable biologic molecules. Previous investigations showed that GT oligonucleotides could exert a specific, dose-dependent cytotoxic effect on human cancer cell lines. This is tightly related to the ability of these oligomers to specifically bind nuclear proteins, giving a complex of apparent molecular weight of 45 kDa. We demonstrated that with respect to the cytotoxic GT-beta-oligomer, alpha-anomeric GT analog did not alter the growth of the T lymphoblastic CCRF-CEM cell line, although the cells took it up efficiently. In agreement with this, GT-alpha-oligomer did not form the cytotoxicity-related 45-kDa complex with nuclear proteins. These findings likely could be related to the ability of GT-alpha to structure under nondenaturing conditions because of the high number of T in the sequence.

Effect of phosphorothioate modifications on the ability of GTn oligodeoxynucleotides to specifically recognize single-stranded DNA-binding proteins and to affect human cancer cellular growth.

We have previously identified phosphodiester oligonucleotides exclusively made of G and T bases, named GTn, that significantly inhibit human cancer cell growth and recognize specific nuclear single-stranded DNA binding proteins. We wished to examine the ability of the modified GTn oligonucleotides with different degrees of phosphorothioate modifications to bind specifically to the same nuclear proteins recognized by the GTn phosphodiester analogues and their cytotoxic effect on the human T-lymphoblastic CCRF-CEM cell line. We showed that the full phosphorothioate GTn oligonucleotide was neither able to specifically recognize those nuclear proteins, nor cytotoxic. In contrast, the 3'-phosphorothioate-protected GTn oligonucleotides can maintain the specific protein-binding activity. The end-modified phosphorothioate oligonucleotides were also able to elicit the dose-dependent cell growth inhibition effect, but a loss in the cytotoxic ability was observed increasing the extent of sulphur modification of the sequences. Our results indicate that phosphorothioate oligonucleotides directed at specific single-stranded DNA-binding proteins should contain a number of phosphorothioate end-linkages which should be related to the length of the sequence, in order to maintain the same biological activities exerted by their phosphodiester analogues.

Formation of triple helices at irregular poly (R.Y) sites located in critical positions in the human bcr promoter.

Two polypurine sequences interrupted respectively by one and two adjacent pyrimidines have been identified in the promoter of the human bcr gene. Although these targets are irregular they are recognised and tightly bound by AG and GT motif triplex-forming oligonucleotides. Thermodynamic and kinetic data are presented.

Effect of oligomer length and base substitutions on the cytotoxic activity and specific nuclear protein recognition of GTn oligonucleotides in the human leukemic CCRF-CEM cell line.

We have identified phosphodiester oligonucleotides composed of G and T bases, named GTn, which are able to inhibit the cellular growth of human cancer cell lines by recognising specific nuclear proteins. We demonstrated that GTn oligonucleotides require a length of at least 20 nucleotides in order to exert a significant cytotoxic effect and to retain the specific protein binding ability. In addition, we found that GTn cytotoxicity was lost when A or C bases were introduced at either 3' and 5' end or within the GTn sequences.

Reduction of mdr1 gene amplification in human multidrug-resistant LoVo DX cell line is promoted by triple helix-forming oligonucleotides.

We have demonstrated previously that the GT triplex-forming oligodeoxyribonucleotide (TFO) d(TGTGTTTTTGTTTTGTTGGTTTTGTTT), named TFO ID, targeted to a polypyrimidine-polypurine coding sequence located within human multidrug-resistance mdrl gene, specifically and significantly reduced mdrl mRNA levels in the drug-resistant T-leukemic CEM-VLB100 cell line. In this article, we demonstrate that TFO 1D is effective at inhibiting not only transcription but also replication of mdrl genes, leading to a loss of amplified gene copies in the drug-resistant colon adenocarcinoma LoVo DX cell line. In contrast, TFO ID does not alter replication of the constitutive mdrl gene copy in the corresponding parental sensitive LoVo 109 cell line. A specific reduction in mdrl gene amplification levels was also obtained with the pyrimidine TFO d(CTTTTTCTTTTCTTCCTTTTCTTT), named TFO 24TC, directed against the same polypyrimidine-polypurine sequence of the mdrl gene. We suggest that triple helix-forming oligonucleotides might affect the replication of unstable chromosomal elements as amplicons in actively replicating cells by causing a local impairment of DNA polymerase activity. This study lends support to the notion that TFO may be used to reduce gene amplification aiming to control neoplastic progression in cancer cells bearing amplified oncogenes.

Effect of cations on purine.purine.pyrimidine triple helix formation in mixed-valence salt solutions.

The effect of various monovalent, divalent and oligovalent cations on the reaction of triplex formation by GT and AG motif triplex-forming oligonucleotides, designed to bind to biologically relevant polypurine-polypyrimidine sequences occurring in the promoters of the murine Ki-ras and human bcr genes, has been investigated by means of electrophoresis mobility shift assays (EMSA) and DNase I footprinting experiments. We found that in the presence of 10 mm MgCl2 the triple helices were progressively destabilized by adding increasing amounts of NaCl, from 20 to 140 mm, to the solution. We also observed that, while the total monovalent-ion concentration was constant at 100 mm, the exchange of sodium with potassium, but not lithium, results in a further destabilization of the triple helices, due to self-association equilibria involving the G-rich triplex-forming oligonucleotides. Potassium was found to destabilize triplex DNA even when the triple helices are preformed in the absence of K+. However, footprinting experiments also showed that the inhibitory effect of K+ on triplex DNA is partially compensated for by millimolar amounts of divalent transition metal ions such as Mn2+ and Ni2+, which upon coordinating to N7 of guanine are expected to enhance hydrogen-bond formation between the target and the third strand, and to reduce the assembly in quadruple structures of G-rich triplex-forming oligonucleotides. Triplex enhancement in the presence of potassium was also observed, but to a lesser extent, when spermine was added to the reaction mixture. Here, the ion effect on triplex DNA is rationalized in terms of competition among the different valence cations to bind to triplex DNA, and differential cation stabilization of unusual quadruplex structures formed by the triplex-forming oligonucleotides.

Human cancer cell lines growth inhibition by GTn oligodeoxyribonucleotides recognizing single-stranded DNA-binding proteins.

Oligonucleotides can specifically target not only nucleic acids but also proteins. Some proteins recognizing oligonucleotides in a sequence-specific manner have been related to cancer transformation and progression. We have found that oligonucleotides composed by repeated and/or variable intervals of GTn with 1 < or = n < or = 7, are able to exert a specific and dose-dependent growth inhibition on human CCRF-CEM, CEM-VLB300, U937, Jurkat, H9 and HeLa tumor cell lines. In contrast, G-->C, G-->A, T-->C and T-->A base substituted control oligonucleotides do not significantly alter cellular growth. In all cell lines, a nuclear protein (molecular mass = 45+/-7 kDa), which specifically recognizes GTn, was identified. Our hypothesis is that the formation of the GTn-protein complex in human cancer cell lines may be involved in the growth inhibition effect. In fact, we found that the reduction or lack of cytotoxic effects by GTn in phorbol 12-myristate 13-acetate-treated CCRF-CEM cells and in normal human lymphocytes is paralleled by the simultaneous reduction or lack of GTn-protein complex. Oligonucleotides specifically 'quenching' intracellular protein activities by forming oligonucleotide-protein complexes may be of potential interest in the treatment of human tumors.

Restoring uptake and retention of daunorubicin and idarubicin in P170-related multidrug resistance cells by low concentration D-verapamil, cyclosporin-A and SDZ PSC 833.

BACKGROUND: Overexpression of the mdr-1 gene that codes for a 170 Kd transmembrane glycoprotein (P170) is a factor responsible for decreased cell sensitivity to anthracyclines and other drugs, and is related to treatment failure in acute leukemia and other tumors. Several agents, including verapamil and cyclosporine derivatives, can modify P170-related resistance in vitro and can be proposed as adjuvant treatment for leukemia and cancer. MATERIALS AND METHODS: To investigate the optimal conditions for adjuvant treatment, D-verapamil (D-VRP), cyclosporin-A (CyA) and the cyclosporine derivative SDZ PSC 833 (PSC) were used alone and in combination at drug concentrations that can be achieved and maintained in vivo. The drugs were tested for their capacity to restore intracellular daunorubicin (DNR) and idarubicin (IDA) accumulation in high-resistance (CEM VLB 300) and intermediate-resistance (CEM VLB 100) cells to levels found in the non-resistant parental cell line (CCRF CEM). RESULTS: In intermediate-resistance cells, IDA alone was more easily restored than DNR plus modifiers, and intracellular IDA concentration was returned to the level of non-resistant cells with low-dose D-VRP (1 microM), CyA (0.8 microM) and PSC (0.4 microM). In high-resistance cells no modifier or modifier combination was able to restore intracellular DNR concentration to the value of non-resistant cells. Intracellular IDA concentration was almost completely restored only with D-VRP (2-3 microM) and CyA (0.8-1.6 microM) in combination or with PSC alone (0.4 microM). CONCLUSIONS: These data suggest that as far as P170-related resistance is concerned, IDA alone is as efficient as or even more efficient than DNR plus modifiers, and that residual resistance to IDA can be overcome with a combination of D-VRP and Cy-A at a clinically achievable concentration, or with a more powerful modifier like SDZ PSC 833.

Effect of unmodified triple helix-forming oligodeoxyribonucleotide targeted to human multidrug-resistance gene mdr1 in MDR cancer cells.

The human mdr1 gene encodes a transmembrane glycoprotein the over-expression of which is associated with development of multidrug resistance in human tumor cells. A negative modulation of human mdr1 has been attempted via a 27-mer unmodified triple helix-forming oligonucleotide, named 1D, targeted to a homopurine sequence in the coding region of the gene. By administering 10 microM of 1D we could find a significant reduction in MDR1 mRNA levels in the human drug-resistant cell line CEM-VLB100. This effect appears to be specific and due to a transient block of RNA polymerase mediated by triple helix formation.

Treatment of sphingomyelinase deficiency by repeated implantations of amniotic epithelial cells.

Five young patients with Niemann-Pick disease type B were treated with repeated implantations of amniotic epithelial cells, as a source of exogenous sphingomyelinase. This treatment abolished the recurrent infections, mainly of the respiratory tract, and led to other improvements of the general conditions of the patients. In particular, we noticed a disappearance of vomiting, a recovery from muscular hypotrophy, and significantly reduced pulmonary distress. In four subjects, who were in a prepuberal state, there was a puberal spurt with a concomitant burst of growth. In two cases, characterized by a greater than normal content of sphingomyelin in urinary sediments, a single implantation caused a sustained normalization of sphingomyelin and total phospholipids in the urine. Finally, sphingomyelinase activity of peripheral leukocytes, when assayed 0.5 to 4 months after some of the implantations, showed a rise to heterozygous values in 30-40% of the assays.

Secretion of lysosomal hydrolases by cultured human amnion epithelial cells.

Primary microcultures of human amnion epithelial cells were established, starting from sterile term placentae. Over a period of 1 week in culture, the epithelial cells release into the extracellular medium substantial amounts of some lysosomal hydrolases, such as sphingomyelinase, N-acetyl-beta-glucosaminidase, alpha-fucosidase, beta-glucuronidase, alpha-mannosidase, and arylsulfatase. Judging from experiments conducted with the protein synthesis inhibitor, cycloheximide, the enzymes released are not newly synthesized forms, but very likely derive from lysosomes. The constitutive secretion of lysosomal enzymes, coupled with lack of immunogenicity, makes amnion epithelial cells a convenient source of enzymes for implantation in attempts of enzyme replacement therapies.

Small, antibacterial and large, inactive peptides of neutrophil granules share immunoreactivity to a monoclonal antibody.

Monoclonal antibodies were raised against a bactericidal protein fraction that was purified from an extract of bovine neutrophil granules and that was previously shown (A. Savoini, R. Marzari, L. Dolzani, D. Serranò, G. Graziosi, R. Gennaro, and D. Romeo, Antimicrob. Agents Chemother. 26:405-407, 1984) to inhibit bacterial DNA synthesis. One of these antibodies, BP97, was covalently linked to Affi-Gel 10 and was used for immunoaffinity chromatography of granule extracts. Elution of the bound proteins, followed by reversed-phase high-performance liquid chromatography, generated several peptides whose molecular weights fell in the range of 1,600 to 64,000 and which reacted to BP97 but not to other mouse monoclonal or polyclonal antibodies. The reaction to BP97 appeared to be specific, inasmuch as a full panel of cationic oligo- or polypeptides was not recognized by this monoclonal antibody. Among the purified granule polypeptides, the more cationic ones (with molecular weights of 4,300 to 8,000) inhibited the growth of Escherichia coli at a MIC of 12 to 50 micrograms/ml. In addition, a 1,600-molecular-weight, highly cationic peptide also inhibited the growth of Staphylococcus aureus and Staphylococcus epidermidis at MICs of 3 to 8 micrograms/ml.

Successful therapy of Niemann-Pick disease by implantation of human amniotic membrane.

In a patient with a lysosomal storage disorder, not involving the CNS, repeated implantations of human amniotic sheets have proved to provide a successful approach to enzyme replacement therapy. Implantation of pure epithelial cells, separated from the other cell types of the amnion, might markedly improve the procedure, avoiding some risks of host-versus-graft rejection.

Modulation of human neutrophil function by C-reactive protein.

Investigation of the effect of C-reactive protein (CRP), an acute-phase reactant, on the functional capacities of human neutrophils was carried out as the basis for elucidating the biological function of C-reactive protein. An initial stimulation at low concentrations, followed by inhibition of superoxide production, and secretion of vitamin-B12-binding protein in the presence of two stimulants, phorbol myristate acetate and concanavalin A, and of neutrophil chemotaxis with increasing concentration of CRP was observed. Correlation between modulation of cell function, at least at relatively high CRP concentrations (greater than 50 micrograms/ml) and an increase in the intracellular level of cAMP is suggested. CRP was also found to enhance neutrophil phagocytosis of particles not containing phosphorylcholine, the native CRP ligand. The proposed role of CRP in neutrophil function is one of regulation and as a negative feedback for potential cytotoxic neutrophil functions.

[Graft of cryopreserved human amniotic epithelial cells in a subject with type B Niemann-Pick disease]. / Impianto di cellule epiteliali amniotiche umane crioconservate in un soggetto affetto da malattia di Niemann-Pick tipo B.

Implantation of pure cryopreserved epithelial cells obtained by enzymatic digestion of human amnion was successfully carried out in one patient affected by Niemann-Pick disease type B. No host-versus-graft reaction was recorded after implantation. The clinical improvement observed in this patient is supposed to be effect of the documented increase of sphingomyelinase activity in his leukocytes after implantation, confirming the possibility that an effective release of sphingomyelinase from amniotic epithelial cells and enzyme uptake by deficient cells can occur. Separation and cryopreservation of human amnion epithelial cells markedly improve the procedures of implantation and may represent a further step beyond in the enzymatic therapy of many lysosomal storage disorders.