Risultati della ricerca
Furocoumarins photolysis products induce differentiation of human erythroid cells.
Viola G , Vedaldi D , Dall'acqua F , Lampronti I , Bianchi N , Zuccato C , Borgatti M , Gambari R .
Department of Pharmaceutical Sciences, University of Padova , Via Marzolo 5, University of Padova , 35131 Padova , Italy .
Psoralens, also known as furocoumarins, are a well-known class of photosensitizers largely used in the therapy of various skin disease. In this study we have evaluated the effects of crude pre-irradiated solutions of furocoumarins derivatives on (a) erythroid differentiation and apoptosis of human leukemic K562 cells and (b) hemoglobin synthesis in cultures of human erythroid progenitors derived from the peripheral blood. To prove the activity of a mixture of photoproducts generated by UVA irradiation of the three psoralen derivatives 5-methoxypsoralen (5-MOP) 8-methoxypsoralen (8-MOP), and angelicin (ANG), we employed the human leukemic K562 cell line and the two-phase liquid culture procedure for growing erythroid progenitors. The results obtained demonstrate that pre-irradiated solutions of psoralen derivatives significantly induce erythroid differentiation of K562 cells irrespective of the type of derivative used, suggesting that the active photoproduct(s) share a common structure. Interestingly, solutions of psoralens irradiated in anaerobic conditions do not exhibits erythroid inducing ability, indicating that the effect is mostly due to photooxidized psoralen products. In erythroid precursor cells, psoralens photolysis products stimulates at low concentrations an increase of hemoglobin A and hemoglobin F. Altogether, these data suggest that photoproducts of psoralen warrant further evaluation as potential therapeutic drugs in beta-thalassaemia and sickle cell anaemia.
2) Apoptosis. 2008 Apr;13(4):553-61.
Human osteoclasts differentiated from umbilical cord blood precursors are less prone to apoptotic stimuli than osteoclasts from peripheral blood.
Penolazzi L , Pocaterra B , Tavanti E , Lambertini E , Vesce F , Gambari R , Piva R .
Molecular Biology Section, Department of Biochemistry and Molecular Biology, Ferrara University, Via Fossato di Mortara, 74, Ferrara, 44100, Italy.
Osteoclasts (OCs) are specialized bone-resorbing cells. For "in vitro" analysis, they may be obtained from the precursors present in peripheral blood (PB) or umbilical cord blood (UCB), but there has been no detailed analysis of how the kind of source and cell culture conditions may affect the behavior of these cells. Here we analyzed the behavior of OCs after transfection with specific transcription factor decoy molecules founding that the OCs from PB undergo apoptosis when nuclear factor kappa B (NF-kB) or NFATc1 were removed, or when ERalpha expression was increased. Conversely, OCs from UCB showed a strong resistance to apoptotic stimuli. We found that survival signals including Bcl-2, Bcl-XL, and Survivin are present in the OCs/UCB, but not in OCs/PB. The resistance to apoptosis seems to be not correlated with NF-kB, NFATc1, or ERalpha expression level, or with the activation of ERK and Akt proteins. One of the mechanisms responsible for bone remodeling is apoptosis, and being susceptible of therapeutic manipulation, the OCs are extensively employed to investigate cell response to therapies for the treatment of bone loss associated with several diseases, including periodontitis, osteoporosis, and metastatic osteolysis. Therefore, our evidences are to be taken in consideration when both the effects of biological modifiers are tested and OCs apoptosis molecular mechanisms are investigated.
3) J Cell Physiol. 2008 Jul;216(1):101-10.
ERalpha and AP-1 interact in vivo with a specific sequence of the F promoter of the human ERalpha gene in osteoblasts.
Lambertini E , Tavanti E , Torreggiani E , Penolazzi L , Gambari R , Piva R .
Department of Biochemistry and Molecular Biology, Molecular Biology Section, University of Ferrara , Ferrara , Italy .
Estrogen-responsive genes often have an estrogen response element (ERE) positioned next to activator protein-1 (AP-1) binding sites. Considering that the interaction between ERE and AP-1 elements has been described for the modulation of bone-specific genes, we investigated the 17-beta-estradiol responsiveness and the role of these cis-elements present in the F promoter of the human estrogen receptor alpha (ERalpha) gene. The F promoter, containing the sequence analyzed here, is one of the multiple promoters of the human ERalpha gene and is the only active promoter in bone tissue. Through electrophoretic mobility shift (EMSA), chromatin immunoprecipitation (ChIP), and re-ChIP assays, we investigated the binding of ERalpha and four members of the AP-1 family (c-Jun, c-fos, Fra-2, and ATF2) to a region located approximately 800 bp upstream of the transcriptional start site of exon F of the human ERalpha gene in SaOS-2 osteoblast-like cells. Reporter gene assay experiments in combination with DNA binding assays demonstrated that F promoter activity is under the control of upstream cis-acting elements which are recognized by specific combinations of ERalpha, c-Jun, c-fos, and ATF2 homo- and heterodimers. Moreover, ChIP and re-ChIP experiments showed that these nuclear factors bind the F promoter in vivo with a simultaneous occupancy stimulated by 17-beta-estradiol. Taken together, our findings support a model in which ERalpha/AP-1 complexes modulate F promoter activity under conditions of 17-beta-estradiol stimulation.
4) Acta Haematol. 2008;119(1):28-37.
A novel frameshift mutation (+A) at codon 18 of the beta-globin gene associated with high persistence of fetal hemoglobin phenotype and deltabeta-thalassemia.
Feriotto G , Salvatori F , Finotti A , Breveglieri G , Venturi M , Zuccato C , Bianchi N , Borgatti M , Lampronti I , Mancini I , Massei F , Favre C , Gambari R .
GenTech-for-Thal, Department of Biochemistry and Molecular Biology, Ferrara University , Ferrara , Italy .
We report in this paper a novel thalassemia mutation (insertion of a single A nucleotide within the exon 1, at codon 18, of the beta-globin gene) associated with a deletion of the deltabeta-globin gene region, in a patient exhibiting high persistence of fetal hemoglobin. The novel mutation causes a frameshift with the generation of a UGA stop codon. Analysis of the parent's DNA demonstrates that the A insertion and frameshift mutation are inherited from the father, while the deltabeta-globin gene deletion is inherited from the mother. Gene dosage analysis and deletion-specific PCR demonstrate that the deletion is the (deltabeta)(0) Sicilian deletion, involving a 13.4-kb deltabeta-globin gene region.
5) Int J Mol Med. 2008 Jan;21(1):3-12.
New trends in non-invasive prenatal diagnosis: applications of dielectrophoresis-based Lab-on-a-chip platforms to the identification and manipulation of rare cells.
Borgatti M , Bianchi N , Mancini I , Feriotto G , Gambari R .
Biotechnology Center and ER-GenTech, Department of Biochemistry and Molecular Biology, University of Ferrara , Ferrara , Italy .
The isolation of rare cells, such as fetal nucleated red blood cells and trophoblasts, from maternal blood for non-invasive prenatal diagnosis is a new field of research exhibiting several difficulties since this strategy requires unresolved basic technological protocols for a successful outcome. However, several achievements in the field of Laboratory-on-a-chip (Lab-on-a-chip) technology have provided clear advancements in projects aimed at the isolation of rare cells from biological fluids. Among the most interesting approaches are those based on dielectrophoresis (DEP). DEP-based Lab-on-a-chip platforms have been demonstrated to be suitable for several applications in biotechnology and biomedicine. DEP-based arrays are able to manipulate single cells, which can be identified and moved throughout the DEP chip to recovery places. DEP buffers are compatible with molecular interactions between monoclonal antibodies and target cells, allowing integration of these devices with magnetic cell sorting (MACS). DEP treatment does not alter the viability of manipulated cells.
6) Biochem Pharmacol. 2008 Feb 15;75(4):810-25. (Traduzione)
Induction of gamma-globin mRNA, erythroid differentiation and apoptosis in UVA-irradiated human erythroid cells in the presence of furocumarin derivatives.
Viola G , Vedaldi D , Dall'Acqua F , Fortunato E , Basso G , Bianchi N , Zuccato C , Borgatti M , Lampronti I , Gambari R .
Department of Pharmaceutical Sciences, University of Padova , Via Marzolo 5, 35131 Padova , Italy . Questo indirizzo e-mail è protetto dallo spam bot. Abilita Javascript per vederlo.
Psoralens, also known as furocoumarins, are a class of photosensitizers largely used in the therapy of various skin diseases. In this study we have evaluated the combined effects of UVA irradiation and furocoumarins derivatives on (a) erythroid differentiation and apoptosis of human leukemia K562 cells and (b) globin gene expression in cultures of human erythroid progenitors derived from the peripheral blood. To prove the activity of a series of linear and angular furocoumarins derivatives, we employed the human leukemia K562 cell line and the two-phase liquid culture procedure for growing erythroid progenitors. Quantitative real-time reverse transcription polymerase-chain assay (Q-RT-PCR) was employed for quantification of the accumulation of globin mRNAs. The results obtained demonstrate that both linear and angular furocoumarins are strong inducers of erythroid differentiation of K562 cells. From a preliminary screening, we have selected two derivatives, 5-methoxypsoralen (5-MOP) and trimethylangelicin (TMA), for which we have investigated their mechanism of action. The cell cycle analysis showed that these derivatives induce, after irradiation, a cell cycle arrest in the G2/M phase, followed by apoptosis. Mitochondrial depolarisation and caspases activation seem to be involved in the mechanism of cell death. In erythroid precursor cells, psoralens in combination with UVA irradiation, stimulate at very low concentrations a preferential increase of gamma-globin mRNA. Altogether, these data suggest that psoralen derivatives warrant further evaluation as potential therapeutic drugs in beta-thalassemia and sickle cell anemia.
7) Cell Biol Int. 2008 Feb;32(2):320-5.
Evaluation of chemokine and cytokine profiles in osteoblast progenitors from umbilical cord blood stem cells by BIO-PLEX technology.
Penolazzi L , Lambertini E , Tavanti E , Torreggiani E , Vesce F , Gambari R , Piva R .
Department of Biochemistry and Molecular Biology, Molecular Biology Section, University of Ferrara, Via Fossato di Mortara, 74, Ferrara, Italy.
We have used cytokine protein array to analyze the secretion of cytokines from an osteoblastic clone derived from human umbilical cord blood mesenchymal stem cells (MSCs) cultured in an osteogenic differentiation medium. The analysis demonstrated the unexpected ability of osteoblast committed cells and their early progenitors to produce significant amounts of a range of soluble immune mediators without in vitro exposure to clinically relevant bacterial pathogens. The cells were expanded and their osteogenic potential analyzed over 45 days of culture was revealed by the expression of osteoblast-specific markers (alkaline phosphatase and Runx2), and by matrix mineralization. Over this culture period, the cells secreted particularly high levels of IL-8, MCP-1 and VEGF, but did not express IL-2, IL-7, IL-17, eotaxin, G-CSF and IFN-gamma. These findings should encourage the use of human umbilical cord blood as a potential stem cells source for bone regeneration.
8) Biopolymers. 2007;88(6):815-22.
Alternate PNA-DNA chimeras (PNA-DNA)(n): synthesis, binding properties and biological activity.
Moggio L , Romanelli A , Gambari R , Bianchi N , Borgatti M , Fabbri E , Mancini I , di Blasio B , Pedone C , Messere A .
Dipartimento di Scienze Ambientali, Seconda Università di Napoli, via Vivaldi 43, 81100 Caserta, Italy.
Peptide nucleic acids (PNAs) are oligonucleotide mimics in which the sugar-phosphate backbone has been replaced by a pseudo-peptide backbone. Among PNA-based molecules, PNA-DNA conjugates characterized by tracts of DNA bound to N and/or C terminus of PNA are very soluble in aqueous media, are able to recognize exclusively single strands of DNA and RNA in antiparallel fashion, activate RNAse H, bind to transcription factors and are more stable than DNA to nucleases degradation. Very little information is available on chimeras constituted of alternating monomers of PNA and DNA. In this article, we describe a simple fully automated strategy for the synthesis of 6-mer and 10-mer alternate PNA-DNA chimeras consisting of polythymine oligomers, stability assays in fetal calf serum, UV and CD studies of the single strand alternate chimeras and of alternate chimera/DNA and alternate chimera/RNA duplexes. Evidences supporting the formation of duplex hybrids were found. Furthermore, the ability of forming Hoogsteen base pairing with duplex DNA was investigated. Finally, we tested the ability of the PNA-DNA alternates in (a) interfering with reverse transcription of eukaryotic mRNA and (b) inhibiting DNA-protein interactions.
9) Mol Pharmacol. 2007 Jun;71(6):1457-62.
Induction of estrogen receptor alpha expression with decoy oligonucleotide targeted to NFATc1 binding sites in osteoblasts.
Penolazzi L , Zennaro M , Lambertini E , Tavanti E , Torreggiani E , Gambari R , Piva R .
Department of Biochemistry and Molecular Biology, Molecular Biology Section, Ferrara , Italy .
The nuclear factor of activated T cell cytoplasmic 1 (NFATc1) is a member of the NFAT family and is strictly implicated in the growth and development of bone. Most studies have focused on the effects of NFATc1 activation on osteoclastogenesis. On the contrary, the specific roles of NFAT in osteoblast differentiation are not well understood and, in some instances, reports of its role are contradictory. In the present study, we demonstrated that NFATc1 was involved in the transcriptional regulation of human estrogen receptor alpha (ERalpha) gene in SaOS-2 osteoblastic like cells. NFATc1 was specifically recruited "in vivo" at C and F distal promoters of ERalpha gene. In addition, it is here identified as the negative transcription factor removed by the RA4-3'decoy oligonucleotide able to induce ERalpha expression in osteoblasts. Ca(2+)/calcineurin-NFAT-mediated signaling pathways and ERalpha-dependent signals are involved in diverse cellular reactions by regulating gene expression under both physiological and pathological conditions. Therefore, our data might be useful for proper manipulation of NFATc1- and ERalpha-mediated cellular reactions in different bone disorders, such as osteoporosis.
10) Exp Cell Res. 2007 May 1;313(8):1548-60.
Human estrogen receptor alpha gene is a target of Runx2 transcription factor in osteoblasts.
Lambertini E , Penolazzi L , Tavanti E , Schincaglia GP , Zennaro M , Gambari R , Piva R .
Department of Biochemistry and Molecular Biology, Molecular Biology Section, Ferrara University, Via Fossato di Mortara, 74, 44100 Ferrara, Italy.
Several studies into the mechanisms involved in control of osteoblast-specific gene expression have identified Runx2 and ERalpha (estrogen receptor alpha) as essential regulators of osteoblast differentiation. Recently, interactions between Runx2 and ERalpha have been described. Here, we investigate the role of Runx2 on the regulation of ERalpha expression by determining its interaction with the F promoter, one of the multiple promoters of the human ERalpha gene and the only one active in bone. We found that, in this promoter, three Runx2-like sites are present. By electrophoretic mobility shift assay in combination with supershift and ChIP experiments, we demonstrated that Runx2 preferentially binds one of the Runx2 motifs of the F promoter. To understand whether or not they are involved in influencing F promoter activity, different promoter-reporter deletion and mutation constructs were transiently transfected into human osteoblastic cells. Comparison of luciferase activities allowed the identification of a prevalent negative role of a sequence context, within the -117,877/-117,426 region, which may be under the control of Runx2 (a) site. Finally, silencing and overexpression of endogenous Runx2 provided evidence that Runx2 has a more complex role than initially expected. In fact, Runx2 (a) and Runx2 (b) sites carried out opposite roles which are conditioned by Runx2 levels in bone cells. Therefore, the resulting F promoter activity may be tightly regulated by a dynamic interplay between these two Runx2 sites, with a predominance of negative effect of the Runx2 (a) site.
11) Curr Med Chem. 2007;14(2):199-212.
Medicinal chemistry of fetal hemoglobin inducers for treatment of beta-thalassemia.
Gambari R , Fibach E .
ER-GenTech, Department of Biochemistry and Molecular Biology, Section of Molecular Biology, University of Ferrara , Ferrara , Italy . Questo indirizzo e-mail è protetto dallo spam bot. Abilita Javascript per vederlo.
In this review we summarize the achievements of medicinal chemistry in the field of pharmacological approaches to the therapy of beta-thalassemia using molecules able to stimulate the production of fetal hemoglobin (HbF). We first describe the molecular basis of the pathology and the biochemical rational of using HbF inducers for therapy; we then outlined the in vitro and in vivo experimental systems suitable for screening of such potential drugs, and finally we describe the different classes of compounds with emphasis on their advantages and disadvantages in the treatment. The results of these reviewed studies indicate that: (a) HbF inducers can be grouped in several classes based on their chemical structure and mechanism of action; (b) clinical trials with some of these inducers demonstrate that they are effective in ameliorating the symptoms of beta-thalassemia; (c) a good correlation was found between HbF stimulation in vivo and in vitro indicating that in vitro testing might be predictive of the in vivo response; (d) combined use of different inducers might maximize the effect, both in vitro and in vivo. However, (e) the response to HbF inducers, evaluated in vitro and in vivo, is variable, and some patients might be refractory to HbF induction by certain inducers; in addition, (f) several considerations call for caution, including the fact that most of the inducers exhibit in vitro cytotoxicity, predicting side effects in vivo following prolonged treatments.
12) Acta Haematol. 2007;117(3):168-76.
Everolimus is a potent inducer of erythroid differentiation and gamma-globin gene expression in human erythroid cells.
Zuccato C , Bianchi N , Borgatti M , Lampronti I , Massei F , Favre C , Gambari R .
GenTech-for-Thal, Department of Biochemistry and Molecular Biology, University of Ferrara , Ferrara , Italy .
We studied the effects of everolimus on the erythroid differentiation of human leukaemic K562 cells and on the cultures of erythroid progenitors derived from the peripheral blood of beta-thalassaemia patients. A quantitative real-time reverse-transcription polymerase chain reaction assay was employed for the quantification of the accumulation of globin mRNAs. The results obtained demonstrate that everolimus is a potent inducer of the erythroid differentiation of K562 cells. Erythroid induction is associated with an increase in alpha- and gamma-globin mRNAs. In erythroid precursor cells from 4 beta-thalassaemia patients, everolimus stimulated a preferential increase (ranging from 1.8- to 7.2-fold) in gamma-globin mRNA. Only minor effects were observed on the expression of alpha-globin genes. These results, in our opinion, are of interest as this compound is already employed in clinical trials as an anti-rejection agent following kidney transplantation. These data suggest that everolimus warrants further evaluation as a potential therapeutic drug in the treatment of beta-thalassaemia.
13) Ann N Y Acad Sci. 2006 Dec;1091:509-16.
Induction of apoptosis of osteoclasts by targeting transcription factors with decoy molecules.
Piva R , Penolazzi L , Zennaro M , Bianchini E , Magri E , Borgatti M , Lampronti I , Lambertini E , Tavanti E , Gambari R .
Department of Biochemistry and Molecular Biology, Section of Molecular Biology, Università degli Studi di Ferrara, Via Fossato di Mortara 74, 44100 Ferrara, Italy.
We review the effects of two transcription factor decoy oligonucleotides on apoptosis of human osteoclasts (OCs). The first decoy molecule was designed to inhibit nuclear factor kappa-B (NF-kappaB) binding to target sequence, the second to increase estrogen receptor (ER) alpha expression. We found that both decoy molecules are potent inducers of apoptosis of human OCs, associated with increase of caspase 3 activity and decrease of interleukin 6 expression. In addition, we provide evidence indicating that these oligonucleotides are active in vivo in inducing OCs apoptosis. Because OCs are essential for skeletal development and remodeling throughout the life of animal and man, the approach described is of potential clinical importance.
14) Int J Mol Med. 2006 Nov;18(5):807-11.
Local in vivo administration of a decoy oligonucleotide targeting NF-kappaB induces apoptosis of osteoclasts after application of orthodontic forces to rat teeth.
Penolazzi L , Magri E , Lambertini E , Calò G , Cozzani M , Siciliani G , Piva R , Gambari R .
ER-GenTech, Department of Biochemistry and Molecular Biology, Molecular Biology Section, University of Ferrara, Ferrara, Italy.
In this study, we report the in vivo effects of a decoy oligonucleotide targeting the nuclear factor kappaB (NF-kappaB) on osteoclasts during forced orthodontic tooth movement in rats. Wistar rats were subjected to orthodontic forces, in the absence or presence of treatment with a decoy molecule mimicking a nonsymmetric NF-kappaB binding site (5'-CGC TGG GGA CTT TCC ACG G-3'). TUNEL staining of fragmented DNA revealed that treatment with NF-kappaB decoy but not with scramble double-stranded oligodeoxynucleotides (ODN) induced a high level of osteoclast apoptosis in vivo. Immunohystochemical analysis for death receptor Fas revealed strong positivity only in samples treated with NF-kappaB decoys, demonstrating that osteoclasts are sensitive to death induction via Fas signaling. Induction of apoptosis in osteoclasts could be a strategy for treatment of excessive osteoclast activity in pathologic conditions such as osteoporosis, peri-articular osteolysis, inflammatory arthritis, Paget's syndrome and tumour-associated osteolytic metastases.
15) Int J Oncol. 2006 Oct;29(4):989-95.
Antiproliferative activity of essential oils derived from plants belonging to the Magnoliophyta division.
Lampronti I , Saab AM , Gambari R .
ER-GenTech, Department of Biochemistry and Molecular Biology, Section of Molecular Biology, University of Ferrara , Ferrara , Italy .
The essential oils obtained from different officinal plants of Lebanon , belonging to the Magnoliophyta division, have been tested for their antiproliferative activity on human erythroleukemic K562 cells. Satureja montana showed the most interesting biological activity in inhibiting the cell growth and inducing erythroid differentiation of K562 cells. The essential oil of Satureja montana was therefore analyzed using a GC/MS (gas chromatography/mass spectrometry) system in order to identify the major constituents and compare them with analysis performed on Satureja hortensis. We demonstrated that the essential oil composition varied with the species, the major constituent of Satureja hortensis being carvacrol (50.61%) and that of Satureja montana being alpha-terpineol (12.66%). In order to identify molecules possibly responsible for the biological activity, commercially available derivatives have been assayed on the K562 cell line. Satureja montana essential oil displayed different natural derivatives characterized by higher activity than those present in Satureja hortensis. The common active principles are alpha-pinene, gamma-terpinene, 4-terpineol, alpha-terpineol, tau-cadinene, tau-cadinol and caryophyllene. Both caryophyllene and alpha-terpineol showed important antiproliferative effects on K562 cells.
16) Eur J Haematol. 2006 Nov;77(5):437-41.
Effects of rapamycin on accumulation of alpha-, beta- and gamma-globin mRNAs in erythroid precursor cells from beta-thalassaemia patients.
Fibach E , Bianchi N , Borgatti M , Zuccato C , Finotti A , Lampronti I , Prus E , Mischiati C , Gambari R .
Department of Haematology, Hadassah - Hebrew University Medical Centre, Jerusalem , Israel .
We studied the effects of rapamycin on cultures of erythroid progenitors derived from the peripheral blood of 10 beta-thalassaemia patients differing widely with respect to their potential to produce foetal haemoglobin (HbF). For this, we employed the two-phase liquid culture procedure for growing erythroid progenitors, high performance liquid chromatography for analysis of HbF production and reverse transcription polymerase chain reaction for quantification of the accumulation of globin mRNAs. The results demonstrated that rapamycin induced an increase of HbF in cultures from all the beta-thalassaemia patients studied and an increase of their overall Hb content/cell. The inducing effect of rapamycin was restricted to gamma-globin mRNA accumulation, being only minor for beta-globin and none for alpha-globin mRNAs. The ability of rapamycin to preferentially increase gamma-globin mRNA content and production of HbF in erythroid precursor cells from beta-thalassaemia patients is of great importance as this agent (also known as sirolimus or rapamune) is already in clinical use as an anti-rejection agent following kidney transplantation. These data suggest that rapamycin warrants further evaluation as a potential therapeutic drug in beta-thalassaemia and sickle cell anaemia.
17) J Med Chem. 2006 Jul 13;49(14):4317-26.
New furan side tetracyclic allopsoralen derivatives: synthesis and photobiological evaluation.
Dalla Via L , Mammi S , Uriarte E , Santana L , Lampronti I , Gambari R , Gia O .
Department of Pharmaceutical Sciences, University of Padova , Via Marzolo 5, 35131 Padova , Italy . Questo indirizzo e-mail è protetto dallo spam bot. Abilita Javascript per vederlo.
Novel tetracyclic allopsoralen derivatives characterized by the condensation of a fourth cyclohexenylic (5-7) or benzenic (8-10) ring at the furan side and a methoxy (5 and 8), a hydroxy (6 and 9), or a dimethylaminopropoxy (7 and 10) side chain in the 10 position of the chromophore were prepared. Compounds 7 and 10 showed a strong photoantiproliferative activity, up to 3 orders of magnitude higher than that of the photochemotherapeutic drug 8-methoxypsoralen (8-MOP). The investigation into the mechanism of action demonstrated for 10 the capacity to intercalate between DNA base pairs in the ground state, to give rise to a covalent photoaddition upon UVA irradiation, and to inhibit polymerase chain reaction (PCR) in a sequence-specific manner. Conversely, compound 7 showed a limited capacity to form an intercalative complex and the lack of ability to photoadd to the macromolecule, thus revealing a novel and unusual behavior for an allopsoralen derivative.
18) Apoptosis. 2005 Oct;10(5):1079-94. (Traduzione)
Induction of apoptosis of human primary osteoclasts treated with a transcription factor decoy mimicking a promoter region of estrogen receptor alpha.
Piva R, Penolazzi L, Lambertini E, Giordano S, Gambari R.
Department of Biochemistry and Molecular Biology, Ferrara University, Ferrara, Italy.
In this paper we investigated how the increase of human estrogen receptor alfa (ERalpha) gene expression may affect breast, osteoblast and osteoclast cells. Increase of ERalpha expression was obtained by interfering with the activity of a negative transcription factor and by removing it with a short and powerful decoy oligonucleotide (RA4-3') mimicking a region of distal promoter C of ERalpha gene. We provide evidence that this decoy was able to induce apoptosis in osteoclasts, but not in osteoblasts and in breast cancer cells, in an estrogen dependent manner. This effect was associated with increase of the levels of Caspase 3 and Fas receptor. Since ERalpha is important in the transcription of different genes and is involved in several pathological processes, including neoplastic and osteopenic diseases, our findings may be of relevance for a possible new therapeutical approach of such diseases.
19) Bioorg Med Chem. 2006 Aug 1;14(15):5204-10.
Effects on erythroid differentiation of platinum(II) complexes of synthetic bile acid derivatives.
Lampronti I , Bianchi N , Zuccato C , Medici A , Bergamini P , Gambari R .
ER-GenTech, Department of Biochemistry and Molecular Biology, Molecular Biology Section, University of Ferrara, Ferrara, Italy.
In this study, we compared some bile acid derivatives and their platinum(II) complexes with respect to their ability to induce erythroid differentiation of human leukemic K562 cells. The complexes analyzed were cis-[(3-dehydrocholanoyliden-L-tartrate)-diammineplatinum(II)] (compound 1) and cis-[di(dehydrocholanoate)-bis(triphenylphosphine)-platinum(II)] (compound 3), together with their free ligands, respectively, 3-dehydrocholanoyliden-L-tartaric acid (compound 2) and dehydrocholanoic acid (4), and their parent compounds, respectively, cisplatin and cis-[dichloride-bis(triphenylphosphine)-platinum(II)] (5). We found that compound 1 stimulates erythroid differentiation of K562 cells and an increase of fetal hemoglobin (HbF) production in erythroid precursor cells isolated from peripheral blood of human subjects. This increase is similar to that obtained by hydroxyurea, a potent inducer of HbF production both in vitro and in vivo. Another important conclusion of this study is related to the evaluation of the effects of compound 1 on production of gamma-globin mRNA in human erythroid precursors grown in the two-stage liquid culture system. We demonstrated that compound 1 induces preferential accumulation of gamma-globin mRNA. The results presented in this manuscript could have practical impact, since it is well known that an increase in HbF production could ameliorate the clinical status of patients with beta-thalassemia and sickle cell anemia.
20) J Biomed Sci. 2005 Oct 14;1-8 [Epub ahead of print] (Traduzione)
N-Arylpiperazine modified analogues of the P2X(7) receptor KN-62 antagonist are potent inducers of apoptosis of human primary osteoblasts.
Penolazzi L, Bianchini E, Lambertini E, Baraldi PG, Romagnoli R, Piva R, Gambari R.
Department of Biochemistry and Molecular Biology, Molecular Biology section, University of Ferrara, 44100, Ferrara, Italy.
The P2X(7) nucleotide receptor is an ATP-gated ion channel that plays an important role in bone cell function. Here, we investigated the effects of L: -tyrosine derivatives 1-3 as potent P2X(7) antagonists on human primary osteoclasts. We found that the level of expression of P2X(7) receptor increased after treatment with the derivatives 1-3, together with the induction of high levels of apoptosis. This effect is associated with activation of caspase-3 and inhibition of expression of IL-6. Interestingly, no pro-apoptotic effect of compounds 1-3 was found on human osteoblasts. Our results suggest that the development of specific P2X(7) receptor antagonists may be considered a useful tool to modulate apoptosis of human osteoclasts. Since bone loss due to osteoclast-mediated resorption represents one of the major unsolved problem in osteopenic disorders, the identification of molecules able to induce apoptosis of osteoclasts is of great interest for the development of novel therapeutic strategies.
21) Med Chem. 2005 Jul;1(4):327-33.
Bangladeshi medicinal plant extracts inhibiting molecular interactions between nuclear factors and target DNA sequences mimicking NF-kappaB binding sites.
Lampronti I , Khan MT , Bianchi N , Ather A , Borgatti M , Vizziello L , Fabbri E , Gambari R
ER-GenTech, Department of Biochemistry and Molecular Biology, Ferrara University , Via L.Borsari, 46, 44100 Ferrara , Italy .
Several medicinal plants can be employed to produce extracts exhibiting biological effects. The aim of this work was to verify the ability of extracts derived from different medicinal plants of Bangladesh in interfering with specific DNA-protein interactions. The rationale for this study is based on the observation that alteration of gene transcription represents a very promising approach to control the expression of selected genes and could be obtained using different molecules acting on the interactions between DNA and transcription factors (TFs). We have analysed the antiproliferative activity of extracts from the medicinal plants Hemidesmus indicus, Polyalthia longifolia, Aphanamixis polystachya, Moringa oleifera, Lagerstroemia speciosa, Paederia foetida, Cassia sophera, Hygrophila auriculata and Ocimum sanctum. Antiproliferative activity was assayed on different human cell lines, including erythroleukemia K562, B-lymphoid Raji, T-lymphoid Jurkat and erythroleukemia HEL cell lines. We employed the electrophoretic mobility shift assay (EMSA) as a suitable technique for the identification of plant extracts altering the binding between transcription factors and the specific DNA elements. We found that low concentrations of Hemidesmus indicus, Polyalthia longifolia, Moringa oleifera and Lagerstroemia speciosa, and very low concentrations of Aphanamixis polystachya extracts inhibit the interactions between nuclear factors and target DNA elements mimicking sequences recognized by the nuclear factor kappaB (NF-kappaB). On the contrary, high amount of extracts from Paederia foetida, Cassia sophera, Hygrophila auriculata or Ocimum sanctum were unable to inhibit NF-kappaB/DNA interactions. Extracts inhibiting both NF-kappaB binding activity and tumor cell growth might be a source for anti-tumor compounds, while extracts inhibiting NF-kappaB/DNA interactions with lower effects on cell growth, could be of interest in the search of compounds active in inflammatory diseases, for which inhibition of NF-kappaB binding activity without toxic effects should be obtained.
22) Int J Mol Med. 2005 Jun;15(6):913-20. (Traduzione)
Separation of white blood cells from erythrocytes on a dielectrophoresis (DEP) based 'Lab-on-a-chip' device.
Borgatti M, Altomare L, Baruffa M, Fabbri E, Breveglieri G, Feriotto G, Manaresi N, Medoro G, Romani A, Tartagni M, Gambari R, Guerrieri R.
Laboratory for the Development of Pharmacological and Pharmacogenomic Therapy of Thalassemia, Biotechnology Center, Italy.
The 'Lab-on-a-chip technology' involves miniaturization of complex analytical procedures and is expected to enable laboratory testing to move from the central laboratory employing complex equipment into non-laboratory settings. We report the application of a printed circuit board (PCB)-based chip, generating dielectrophoretic (DEP)-based cylinder-shaped cages for separation and recovery of white blood cells from erythrocytes. This possibility is of interest to develop low-cost Lab-on-a-chip devices for diagnostic purposes. Accordingly, we demonstrate that white blood cells recovered from this Lab-on-a-chip device are suitable for PCR-based molecular diagnosis procedures employing DNA sequencing or biospecific interaction analysis using surface plasmon resonance and biosensor technology.
23) Br J Haematol. 2004 Aug;126(4):612-21. (Traduzione)
Rapamycin-mediated induction of gamma-globin mRNA accumulation in human erythroid cells.
Mischiati C, Sereni A, Lampronti I, Bianchi N, Borgatti M, Prus E, Fibach E, Gambari R.
Department of Biochemistry and Molecular Biology, University of Ferrara, Ferrara, Italy.
The present study aimed to determine whether rapamycin could increase the expression of gamma-globin genes in human erythroid cells. Rapamycin is a macrocyclic lactone that possesses immunosuppressive, antifungal and anti-tumour properties. This molecule is approved as an immunosuppressive agent for preventing rejection in patients receiving organ transplantation. To verify the activity of rapamycin, we employed two experimental cell systems, the human leukaemia K562 cell line and the two-phase liquid culture of human erythroid progenitors isolated from normal donors and patients with beta-thalassaemia. The results suggested that rapamycin, when compared with cytosine arabinoside, mithramycin and cisplatin, is a powerful inducer of erythroid differentiation and gamma-globin mRNA accumulation in human leukaemia K562 cells. In addition, when normal human erythroid precursors were cultured in the presence of rapamycin, gamma-globin mRNA accumulation and fetal haemoglobin (HbF) production increased to levels that were higher than those obtained using hydroxyurea. These effects were not associated with inhibition of cell growth. Furthermore, rapamycin was found to increase HbF content in erythroid precursor cells from four beta-thalassaemia patients. These results could have practical relevance, because pharmacologically mediated regulation of the expression of human gamma-globin genes, leading to increased HbF, is considered a potential therapeutic approach in haematological disorders, including beta-thalassaemia and sickle cell anaemia.
24) Int J Mol Med. 2004 Aug;14(2):145-52. (Traduzione)
Peptide nucleic acid-DNA decoy chimeras targeting NF-kappaB transcription factors: Induction of apoptosis in human primary osteoclasts.
Penolazzi L, Borgatti M, Lambertini E, Mischiati C, Finotti A, Romanelli A, Saviano M, Pedone C, Piva R, Gambari R.
Department of Biochemistry and Molecular Biology, University of Ferrara, Ferrara, Italy.
Peptide nucleic acids (PNAs) are DNA mimics constituted by a pseudopeptide backbone composed of N-(2-aminoethyl)glycine units. PNAs hybridize with high affinity to complementary sequences of single-stranded RNA and DNA, forming Watson-Crick double helices and are resistant to both nucleases and proteases. While applications of PNAs as antisense and antigene molecules have been described, PNA/DNA and PNA/PNA hybrids are not useful for transcription factor decoy (TFD) pharmacotherapy. By contrast, PNA-DNA-PNA (PDP) chimeras, constituted of sequential PNA, DNA and PNA stretches, are potent decoy molecules in vitro. Interestingly, PDP-based decoys a) are more soluble than PNAs, b) are more resistant than synthetic oligonucleotides to enzymatic activity present in cellular extracts and serum and c) can be delivered with liposomes. In the present study we demonstrated that double-stranded PNA-DNA-PNA chimeras targeting NF-kappaB transcription factors induce apoptosis of human primary osteoclasts. Our data suggest that PDP-based induction of osteoclast apoptosis could be a therapeutic approach for disorders in which bone resorption is inappropriately excessive.
25) Mol Diagn. 2004;8(1):33-41. (Traduzione)
Surface plasmon resonance and biosensor technology for real-time molecular diagnosis of beta o 39 thalassemia mutation.
Feriotto G, Breveglieri G, Gardenghi S, Carandina G, Gambari R.
Laboratory for the Development of Pharmacological and Pharmacogenomic Treatment of Thalassemia, Biotechnology Center, Ferrara University, Ferrara, Italy.
BACKGROUND: Biospecific interaction analysis (BIA) employing surface plasmon resonance (SPR) and biosensor technologies is of interest in clinical genetics. However, few data are available on its use in hereditary diseases caused by genetic mutations. AIM: The primary aim of this study was the refinement of BIA technology for use in identifying the beta o 39 mutation of the beta-globin gene, a mutation which causes a common type of beta o thalassemia. METHODS: Target-biotinylated PCR products were immobilized on streptavidin-coated sensor chips and diagnosed using SPR-based BIA performed by injecting specific oligonucleotide probes into the sensor chip. RESULTS: We demonstrated that the beta o 39 mutation can be easily and reproducibly identified during the association phase. CONCLUSIONS: This should be considered a pilot study demonstrating the ability of SPR-based BIA to detect point mutations in the beta-globin gene by real-time monitoring of hybridization between oligonucleotide probes and target-biotinylated PCR products generated from genomic DNA from normal, heterozygous individuals and homozygous beta o thalassemia patients.
26) Lab Invest. 2004 Jun;84(6):796-803. (Traduzione)
Real-time multiplex analysis of four beta-thalassemia mutations employing surface plasmon resonance and biosensor technology.
Feriotto G, Breveglieri G, Finotti A, Gardenghi S, Gambari R.
Laboratory for the Development of Pharmacological and Pharmacogenomic Therapy of Thalassemia, Biotechnology Center, Ferrara University, Ferrara, Italy.
In this paper, biospecific interaction analysis (BIA) employing surface plasmon resonance (SPR) and biosensor technologies was applied to the analysis of multiple mutations of the human beta-globin gene. To this aim, large target polymerase chain reaction (PCR) products were immobilized on sensor chips and then probes detecting beta degrees 39 (C>T), beta degrees IVSI-1 (G>A), beta(+)IVSI-6 (T>C) and beta(+)IVSI-110 (G>A) thalassemia mutations were sequentially injected. In this study, a total of ten normal and seven heterozygous subjects, and six homozygous patients were considered. The results obtained allow to conclude that discrimination between normal subjects, heterozygous, and homozygous patients is readily achieved for all the four mutations by PCR amplification of genomic DNA containing all the regions corresponding to the same mutations, immobilization of the same PCR products, and hybridization. To our knowledge the procedure described here is the first reported on the use of SPR-based BIA and biosensor technology for multiple detections of point mutations.
27) Biochem Pharmacol. 2003 Oct 1;66(7):1189-98. (Traduzione)
Decoy oligodeoxynucleotides targeting NF-kappaB transcription factors: induction of apoptosis in human primary osteoclasts.
Penolazzi L, Lambertini E, Borgatti M, Piva R, Cozzani M, Giovannini I, Naccari R, Siciliani G, Gambari R.
Department of Biochemistry and Molecular Biology, University of Ferrara, Ferrara, Italy.
Proteins belonging to the nuclear factor kappaB (NF-kappaB) superfamily are involved in osteoclast formation, playing a very important role for both differentiation of osteoclast precursors and survival of mature osteoclasts. Several drugs used to fight bone loss in a variety of human pathologies, including osteoporosis, act by increasing the frequency of osteoclast apoptosis, since it was demonstrated that small changes in osteoclast apoptosis can result in large changes in bone formation. In this respect, targeting of NF-kappaB transcription factor could be of great interest. Among nonviral gene therapy strategies recently proposed to inhibit or even block NF-kappaB activity, the transcription factor decoy (TFD) should be taken in great consideration. The main issue of the present study was to examine the effects of decoy DNA/DNA molecules targeting NF-kappaB on apoptosis of human osteoclasts (OCs), with the aim to interfere with the pathway regulating osteoclast differentiation and programmed cell death. To this aim, we used a mixture of receptor activator of NF-kappaB ligand (RANKL), macrophage colony-stimulating factor (M-CSF) and parathyroid hormone (PTH) to prepare human OCs from peripheral blood cells. Then, transfection with the decoy molecules targeting NF-kappaB was performed. The results obtained demonstrate that in primary cells expressing typical osteoclast markers such as TRAP and MMP9, NF-kappaB decoy significantly stimulated apoptosis. Inhibition of IL-6 expression and induction of Caspase 3 were found in OCs treated with NF-kappaB DNA/DNA decoys. We consider these data as the basis for setting up experimental conditions allowing nonviral gene therapy of several bone disorders.
28) Eur J Haematol. 2003 Sep;71(3):189-95. (Traduzione)
Accumulation of gamma-globin mRNA in human erythroid cells treated with angelicin.
Lampronti I, Bianchi N, Borgatti M, Fibach E, Prus E, Gambari R.
Department of Biochemistry and Molecular Biology, University of Ferrara, Ferrara, Italy.
The aim of the present study was to determine whether angelicin is able to increase the expression of gamma-globin genes in human erythroid cells. Angelicin is structurally related to psoralens, a well-known chemical class of photosensitizers used for their antiproliferative activity in treatment of different skin diseases (i.e., psoriasis and vitiligo). To verify the activity of angelicin, we employed two experimental cell systems, the human leukemic K562 cell line and the two-phase liquid culture of human erythroid progenitors isolated from normal donors. The results of our investigation suggest that angelicin, compared with cytosine arabinoside, mithramycin and cisplatin, is a powerful inducer of erythroid differentiation and gamma-globin mRNA accumulation of human leukemia K562 cells. In addition, when normal human erythroid precursors were cultured in the presence of angelicin, increases of gamma-globin mRNA accumulation and fetal hemoglobin (HbF) production, even higher than those obtained using hydroxyurea, were detected. These results could have practical relevance, as pharmacologically-mediated regulation of the expression of human gamma-globin genes, leading to HbF induction, is considered a potential therapeutic approach in hematological disorders, including beta-thalassemia and sickle cell anemia.
29) Blood. 2003 Aug 15;102(4):1276-81.
Mithramycin induces fetal hemoglobin production in normal and thalassemic human erythroid precursor cells.
Fibach E, Bianchi N, Borgatti M, Prus E, Gambari R.
Department of Hematology, Hadassah University Hospital, Jerusalem, Israel; Department of Biochemistry and Molecular Biology, University of Ferrara, Ferrara, Italy.
We report in this paper that the DNA-binding drug mithramycin is a potent inducer of gamma-globin mRNA accumulation and fetal hemoglobin (HbF) production in erythroid cells from healthy human subjects and beta-thalassemia patients. Erythroid precursors derived from peripheral blood were grown in 2-phase liquid culture. In this procedure, early erythroid progenitors proliferate and differentiate during phase 1 (in the absence of erythropoietin) into late progenitors. In phase 2, in the presence of erythropoietin, the latter cells continue their proliferation and mature into Hb-containing orthochromatic normoblasts. Compounds were added on days 4 to 5 of phase 2 (when cells started to synthesize Hb), and cells were harvested on day 12. Accumulation of mRNAs for gamma-globin, beta-globin, alpha-globin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and beta-actin were measured by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR); induction of HbF was analyzed by high-performance liquid chromatography (HPLC) and, at cellular level, by flow cytometry. We demonstrated that mithramycin was able to up-regulate preferentially gamma-globin mRNA production and to increase HbF accumulation, the percentage of HbF-containing cells, and their HbF content. Mithramycin was more effective than hydroxyurea, being, in addition, not cytotoxic. This was shown by the lack of cytotoxicity on erythroid and myeloid in vitro primary cell cultures treated with mithramycin at concentrations effective for HbF induction. These results are of potential clinical significance because an increase of HbF alleviates the symptoms underlying beta-thalassemia and sickle cell anemia. The results of this report suggest that mithramycin and its analogs warrant further evaluation as potential therapeutic drugs.
30) Bioorg Med Chem. 2002 Feb;10(2):347-53. (Traduzione)
Preparation and evaluation of the in vitro erythroid differentiation induction properties of some esters of methyl 3,4-O-isopropylidene-beta-D-galactopyranoside and 2,3-O-isopropylidene-D-mannofuranose.
Catelani G, D'Andrea F, Mastrorilli E, Bianchi N, Chiarabelli C, Borgatti M, Martello D, Gambari R.
Dipartimento di Chimica Bioorganica e Biofarmacia, Universita di Pisa, Via Bonanno, 33-56126, Pisa, Italy; Department of Biochemistry and Molecular Biology, University of Ferrara, Ferrara, Italy.
Two series of glycide esters of short fatty acids, designed for avoiding intramolecular transesterification, were prepared and tested for in vitro erythroid differentiation induction activities using the K562 cell line as experimental system. The 6-O-isobutiryl and pivaloyl derivatives of methyl 3,4-O-isopropylidene-beta-D-galactopyranosides as well the same 1-O-esters of 2,3-O-isopropylidene-alpha- and beta-D-mannofuranose exhibit biological activities much higher that the corresponding acids and could be proposed as possible agents to modulate production of embryo-fetal hemoglobins by human erythroid cells.
31) Am J Pharmacogenomics 2001;1(2):119-35 (Traduzione)
Biospecific interaction analysis: a tool for drug discovery and development.
Gambari R.
Department of Biochemistry and Molecular Biology, and Biotechnology Center, Ferrara University, Ferrara, Italy. Questo indirizzo e-mail è protetto dallo spam bot. Abilita Javascript per vederlo.
The recent development of surface plasmon resonance (SPR)-based biosensor technologies for biospecific interaction analysis (BIA) enables the monitoring of a variety of molecular reactions in real-time. The biomolecular interactions occur at the surface of a flow cell of a sensor chip between a ligand immobilized on the surface and an injected analyte. SPR-based BIA offers many advantages over most of the other methodologies available for the study of biomolecular interactions, including full automation, no requirement for labeling, and the availability of a large variety of activated sensor chips that allow immobilization of DNA, RNA, proteins, peptides and cells. The assay is rapid and requires only small quantitities of both ligand and analyte in order to obtain informative results. In addition, the sensor chip can be re-used many times, leading to low running costs. Aside from the analysis of all possible combinations of peptide, protein, DNA and RNA interactions, this technology can also be used for screening of monoclonal antibodies and epitope mapping, analysis of interactions between low molecular weight compounds and proteins or nucleic acids, interactions between cells and ligands, and real-time monitoring of gene expression. Applications of SPR-based BIA in medicine include the molecular diagnosis of viral infections and genetic diseases caused by point mutations. Future perspectives include the combinations of SPR-based BIA with mass spectrometry, the use of biosensors in proteomics, and the application of this technology to design and develop efficient drug delivery systems.
32) Curr Pharm Des 2001, 7(17):1839-62 (Traduzione)
Peptide-nucleic acids (PNAs): a tool for the development of gene expression modifiers.
Gambari R.
Department of Biochemistry and Molecular Biology, Via L. Borsari n.46, 44100 Ferrara, Italy. Questo indirizzo e-mail è protetto dallo spam bot. Abilita Javascript per vederlo.
Peptide nucleic acids (PNAs) represent nucleic acid analogues with unique biochemical properties and of great interest for the development of therapeutic agents. The firstly designed and tested PNAs are molecules in which the sugar-phosphate backbone of DNA was replaced with a pseudopeptide chain constituted by N-(2-aminoethyl) glycine monomers. Nucleobases can be linked to this backbone through a carboxymethyl moiety, which allows to maintain a two atom spacer between the backbone and the bases. Since the first reports on PNAs based on N-(2-aminoethyl) glycine backbone, other PNA analogues have been synthesized, with the main purpose of improve biological activities as well as stability and efficient delivery to target cells. Of great interest are chiral PNAs, PNA analogues bearing phosphate groups (PHONA),PNA-DNA and PNA-peptide chimeras, PNA linked to non-peptide vectors. PNAs hybridize to DNA and RNA with high efficiency following the Watson-Crick hybridization rules, forming highly stable PNA/DNA and PNA/RNA duplexes. In addition, homopyrimidine PNAs, as well as PNAs containing a high pyrimidine:purine ratio, are able to bind to DNA or RNA forming highly stable (PNA)(2)-DNA triple helices. Accordingly, therapeutic PNA and PNA analogues could act as antigene as well as antisense molecules. In addition, recent studies provide evidences for the possible use of PNA-based therapeutic molecules as artificial promoters, as decoy or ribozyme facilitator. Among the therapeutic applications of PNA-based molecules, the most promising include anti-cancer and anti-viral experimental strategies, but activity of PNAs against bacteria and medically important parasitic organisms have been also reported.
33) Br J Haematol 2001, 113(4):951-61 (Traduzione)
Accumulation of gamma-globin mRNA and induction of erythroid differentiation after treatment of human leukaemic K562 cells with tallimustine.
Bianchi N, Chiarabelli C, Borgatti M, Mischiati C, Fibach E, Gambari R.
Department of Biochemistry and Molecular Biology, University of Ferrara, Italy.
Human leukaemic K562 cells can be induced in vitro to erythroid differentiation by a variety of chemical compounds, including haemin, butyric acid, 5-azacytidine, cytosine arabinoside, mithramycin and chromomycin, cisplatin and cisplatin analogues. Differentiation of K562 cells is associated with an increase of expression of embryo-fetal globin genes, such as the zeta-, epsilon- and gamma-globin genes. The K562 cell line has been proposed as a very 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 stimulating gamma-globin synthesis could be considered for possible use in the therapy of haematological diseases associated with a failure in the expression of normal beta-globin genes. We have analysed the effects of tallimustine and distamycin on cell growth and differentiation of K562 cells. The results demonstrated that tallimustine is a potent inducer, while distamycin is a weak inducer, of K562 cell erythroid differentiation. Erythroid differentiation was associated with an increase of accumulation of gamma-globin mRNA and of production of both haemoglobin (Hb) Gower 1 and Hb Portland. In addition, tallimustine-mediated erythroid induction occurred in the presence of activation of the apoptotic pathway. The reasons for proposing tallimustine as an inducer of gamma-globin gene expression are strongly sustained by the finding that this compound stimulates fetal haemoglobin production in human erythroid precursor cells from normal subjects.
34) Biochem Pharmacol 2000, 60(1):31-40 (Traduzione)
Induction of erythroid differentiation of human K562 cells by cisplatin analogs.
Bianchi N, Ongaro F, Chiarabelli C, Gualandi L, Mischiati C, Bergamini P, Gambari R.
Departments of Biochemistry and Molecular Biology, University of Ferrara, 44100, Ferrara, Italy.
Human leukemic K562 cells can be induced in vitro to erythroid differentiation by a variety of chemical compounds, including hemin, butyric acid, 5-azacytidine, and cytosine arabinoside. Differentiation of K562 cells is associated with an increase in the 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 very useful in vitro model system for determining the therapeutic potential of new differentiating compounds as well as for studying the molecular mechanism(s) regulating changes in the expression of embryonic and fetal human globin genes. Inducers of erythroid differentiation that stimulate gamma-globin synthesis could be considered for possible use in the experimental therapy of hematological diseases associated with a failure in the expression of adult beta-globin genes. In this paper, we analyzed the effects of a series of cisplatin analogs on both cell growth and differentiation of K562 cells. Among seven cisplatin analogs studied, three were found to be potent inducers of erythroid differentiation. Erythroid differentiation was associated with an increase in the accumulation of (a) hemoglobins Gower 1 and Portland and (b) gamma-globin mRNA.
35) Bioorg Med Chem Lett 1999; 9(21):3153-8 (Traduzione)
Induction of erythroid differentiation of human K562 cells by 3-O-acyl-1,2-O-isopropylidene-D-glucofuranose derivatives.
Catelani G, Osti F, Bianchi N, Bergonzi MC, D'Andrea F, Gambari R.
Dipartimento di Chimica Bioorganica e Biofarmacia, Universita di Pisa, Italy.
In this paper we report the synthesis of twelve 3-O-acyl-1,2-O-isopropylidene-D-glucofuranose derivatives and the results obtained on their effects in inducing erythroid differentiation of human leukemic K562 cells. The data obtained demonstrate that two of the newly synthetized compounds are able to induce erythroid differentiation of K562 cells. In addition, these same compounds potentiate K562 erythroid differentiation induced by cytosine arabinoside, retinoic acid and mithramycin. Inducers of erythroid differentiation stimulating fetal gamma-globin synthesis could be considered for possible use in the experimental therapy of hematological diseases associated with a failure in the expression of adult beta-globin genes.
36) Am J Hematol 1999, 62(1):33-43 (Traduzione)
In vitro effect on human leukemic K562 cells of co-administration of liposome-associated retinoids and cytosine arabinoside (Ara-C).
Cortesi R, Gui V, Gambari R, Nastruzzi C.
Department of Pharmaceutical Sciences, University of Ferrara, Via L. Borsari 46, 44100 Ferrara, Italy.
The administration of retinoids has been demonstrated to be of potential utility in the therapy of a wide spectrum of neoplastic pathologies due to the ability to induce differentiation in a large variety of primary tumor cells as well as in vitro cultured cell lines. Moreover, a number of compounds, including hemin, cytosine arabinoside, and 5-azacytidine are able to induce erythroid differentiation of the erythroleukemic cell line K562. In this paper we determined whether a combined treatment of K562 cells with suboptimal concentrations of cytosine arabinoside and retinoids containing liposomes lead to a full expression of differentiated functions. Liposomes were prepared by reverse phase evaporation technique followed by extrusion through polycarbonate filters. Cell growth kinetics studies and intracellular detection of hemoglobin by benzidine staining were performed. The results obtained showed that the combined treatment with liposomes containing retinoids and sub-optimal concentration of ara-C is an effective strategy to induce K562 cell differentiation, minimizing at the same time toxic effects. Control experiments aimed to determine possible selection of subpopulations of K562 cells suggest that the observed results are not related to toxicity and/or potential selection of induced cells. In conclusion, liposomally delivered retinoids could be proposed for differentiation therapy as an effective strategy in the treatment and management of malignancy. In addition, the finding that liposomally delivered retinoids increase the capacity of cytosine arabinoside to induce erythroid differentiation, could be of interest in studies aimed at the development of treatment able to reactivate fetal globin genes in beta-thalassemia patients.
37) Br J Haematol 1999, 104(2):258-65 (Traduzione)
The DNA-binding drugs mithramycin and chromomycin are powerful inducers of erythroid differentiation of human K562 cells.
Bianchi N, Osti F, Rutigliano C, Corradini FG, Borsetti E, Tomassetti M, Mischiati C, Feriotto G, Gambari R.
Department of Biochemistry and Molecular Biology, University of Ferrara, Italy.
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.
38) Eur J Haematol 1998, 61(5):295-301 (Traduzione)
Human leukemic K562 cells treated with cytosine arabinoside: enhancement of erythroid differentiation by retinoic acid and retinol.
Cortesi R, Gui V, Osti F, Nastruzzi C, Gambari R.
Department of Pharmaceutical Sciences, University of Ferrara, Italy.
Human leukemia K562 cells can be induced to erythroid differentiation when treated with a variety of compounds, including hemin, cytosine arabinoside and 5-azacytidine. Following erythroid induction, K562 cells express at high level gamma-globin and accumulate both Hb Portland and Hb Gower 1. In this paper we determined whether a combination treatment of K562 cells with suboptimal concentrations of cytosine arabinoside and retinoids lead to full expression of differentiated functions. Cell growth kinetics studies, intracellular detection of hemoglobin by benzidine staining and hemoglobin analysis by cellulose acetate were performed. The results obtained show that (a) retinoic acid and retinol are not able to induce differentiation of K562 cells and (b) cytosine arabinoside induces differentiation only when used at 100-300 nmol/l concentrations. In addition, our data demonstrate that erythroid differentiation of K562 occurs when 40 micromol/l of retinoic acid or retinol are added together with 75 nmol/l cytosine arabinoside.
39) Haematologica 1997, 82(4):395-401 (Traduzione)
Human leukemia K562 cells: induction to erythroid differentiation by guanine, guanosine and guanine nucleotides.
Osti F, Corradini FG, Hanau S, Matteuzzi M, Gambari R.
Department of Biochemistry and Molecular Biology, University of Ferrara, Italy.
BACKGROUND AND OBJECTIVE: Human leukemic K562 cells are able to undergo erythroid differentiation in vitro when cultured with a variety of inducers, leading to increased expression of embryo-fetal globin genes such as the zita, epsilon and gamma-globin genes. Therefore the K562 cell line has been proposed as a very useful in vitro model system for determining the therapeutical potential of new differentiating compounds as well as for studying the molecular mechanism(s) that regulate changes in the expression of embryonic and fetal human globin genes. In this study we explored whether nucleoside triphosphates and related compounds are able to induce differentiation of K562 cells. METHODS: K562 cell differentiation was studied using the benzidine test; hemoglobins were characterized by cellulose acetate gel electrophoresis and mRNA accumulation was investigated by Northern blot analysis. RESULTS: The main conclusion of this paper is that guanine, guanosine and guanine ribonucleotides are effective inducers of K562 cell differentiation. Expression of both Hb Portland and Hb Gower 1 is increased in GTP-induced K562 cells. This increase is associated with greater gamma-globin mRNA accumulation. By contrast, ATP, CTP and UTP are not able to induce erythroid differentiation. INTERPRETATION AND CONCLUSIONS: These findings suggest that guanine, guanosine and guanine ribonucleotides are inducers of erythroid differentiation of K562 cells. This is of some relevance since differentiating compounds have been proposed as antitumor agents. In addition, inducers of erythroid differentiation that stimulate gamma-globin synthesis might be considered in the experimental therapy of hematological diseases associated with a failure in the expression of adult beta-globin genes.
