Carlo Mischiati

Induction of erythroid differentiation of human K562 cells by cisplatin analogs

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.

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 ζ, ε and γ 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 γ‐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 β‐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) γ‐globin mRNA.

Accumulation of γ‐globin mRNA and induction of erythroid differentiation after treatment of human leukaemic K562 cells with tallimustine

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 ζ‐, ε‐ and γ‐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 γ‐globin synthesis could be considered for possible use in the therapy of haematological diseases associated with a failure in the expression of normal β‐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 γ‐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 γ‐globin gene expression are strongly sustained by the finding that this compound stimulates fetal haemoglobin production in human erythroid precursor cells from normal subjects.

Transcription Factor Decoy Molecules Based on a Peptide Nucleic Acid (PNA)-DNA Chimera Mimicking Sp1 Binding Sites

Peptide nucleic acids (PNAs) are DNA-mimicking molecules in which the sugar-phosphate backbone is replaced by a pseudopeptide backbone composed of N-(2-aminoethyl)glycine units. We determined whether double-stranded molecules based on PNAs and PNA-DNA-PNA (PDP) chimeras could be capable of stable interactions with nuclear proteins belonging to the Sp1 transcription factor family and, therefore, could act as decoy reagents able to inhibit molecular interactions between Sp1 and DNA. Since the structure of PNA/PNA hybrids is very different from that of the DNA/DNA double helix, they could theoretically alter the molecular structure of the double-stranded PNA-DNA-PNA chimeras, perturbing interactions with specific transcription factors. We found that PNA-based hybrids do not inhibit Sp1/DNA interactions. In contrast, hybrid molecules based on PNA-DNA-PNA chimeras are very effective decoy molecules, encouraging further experiments focused on the possible use of these molecules for the development of potential agents for a decoy approach in gene therapy. In this respect, the finding that PDP-based decoy molecules are more resistant than DNA/DNA hybrids to enzymatic degradation appears to be of great interest. Furthermore, their resistance can even be improved after complexation with cationic liposomes to which PDP/PDP chimeras are able to bind by virtue of their internal DNA structure.

Interaction of the Human NF-κB p52 Transcription Factor with DNA-PNA Hybrids Mimicking the NF-κB Binding Sites of the Human Immunodeficiency Virus Type 1 Promoter

We determined whether peptide nucleic acids (PNAs) are able to interact with NF-κB p52 transcription factor. The binding of NF-κB p52 to DNA-DNA, DNA-PNA, PNA-DNA, and PNA-PNA hybrid molecules carrying the NF-κB 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-κB p52 does not efficiently bind to PNA-PNA hybrids. However, a DNA-PNA hybrid molecule was found to be recognized by NF-κB p52, although the molecular complexes generated exhibited low stability. From the theoretical point of view, our results suggest that binding of NF-κB 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-κB p52 protein, although with an efficiency lower than DNA-DNA NF-κB target molecules; therefore, our results should encourage studies on modified PNAs in order to develop potential agents for the decoy approach in gene therapy.

Liposomes as carriers for DNA–PNA hybrids

Peptide nucleic acids (PNAs) are DNA mimics composed of N-(2-aminoethyl)glycine units. This structure gives to PNAs (a) resistance to DNases and proteinases, (b) capacity to hybridize with high affinity to complementary sequences of single-stranded RNA and DNA, and (c) capacity to form highly stable (PNA)(2)-RNA triplexes with RNA targets. Furthermore, DNA-PNA hybrid molecules are capable to reversibly interact with DNA-binding proteins, being therefore of interest for studies on regulation of gene expression by the decoy approach. The major conclusion of this paper is that cationic liposomes are able to efficiently complexate DNA-PNA hybrid molecules and mediate their binding to target cells. Our results are of some interest, since, unlike commonly used nucleic acids analogs, PNA oligomers are not taken up spontaneously into the cells. In addition, they are not suitable for an efficient delivery with commonly used liposomal formulations. Transfection of PNA-DNA hybrid molecules to in vitro cultured cells could be of great interest to determine the applications of these new reagents to experimental alteration of gene expression.

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.

Effects of rapamycin on accumulation of α-, β- and γ-globin mRNAs in erythroid precursor cells from β-thalassaemia patients

Abstract:  We studied the effects of rapamycin on cultures of erythroid progenitors derived from the peripheral blood of 10 β‐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 β‐thalassaemia patients studied and an increase of their overall Hb content/cell. The inducing effect of rapamycin was restricted to γ‐globin mRNA accumulation, being only minor for β‐globin and none for α‐globin mRNAs. The ability of rapamycin to preferentially increase γ‐globin mRNA content and production of HbF in erythroid precursor cells from β‐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 β‐thalassaemia and sickle cell anaemia.

Effects of a Hydroxyapatite-based Biomaterial on Gene Expression in Osteoblast-like Cells

Biostite® is a hydroxyapatite-derived biomaterial that is used in periodontal and bone reconstructive procedures due to its osteoconductive properties. Since the molecular effects of this biomaterial on osteoblasts are still unknown, we decided to assess whether it may specifically modulate osteoblast functions in vitro. We found that a brief exposure to Biostite® significantly reduced the proliferation of MG-63 and SaOS-2 osteoblast-like cells to ~ 50% of the plateau value. Furthermore, gene array analysis of MG-63 cells showed that Biostite® caused a differential expression of 37 genes which are involved in cell proliferation and interaction, and related to osteoblast differentiation and tissue regeneration. Results were confirmed by RT-PCR, Western blot, and by an increase in alkaline phosphatase (ALP) specific activity. Biostite® also increased levels of polycystin-2, a mechano-sensitive Ca2+ channel, a promising new marker of bone cell differentiation. Biostite®, therefore, may directly affect osteoblasts by enhancing chondro/osteogenic gene expression and cytoskeleton-related signaling pathways, which may contribute to its clinical efficacy.

Extracellular HIV-1 Tat protein differentially activates the JNK and ERK/MAPK pathways in CD4 T cells.

OBJECTIVE To investigate the intracellular signals elicited by extracellular HIV-1 Tat protein in lymphoid CD4 T cells. METHODS CD4 Jurkat T cells were treated with a series of glutathione S-transferase (GST)-Tat fusion proteins: full-length two-exon GST-Tat (GST-Tat2E); one-exon Tat, in which the second exon of Tat was deleted (GST-Tat1E); two-exon Tat, in which the seven arginine residues have been changed to alanine residues (GST-TatArg(mut)), GST-TatdeltaN, which shows a deletion of the N-terminal 21 amino acids. The cells were either treated with soluble GST-Tat proteins or seeded on plates coated with GST-Tat proteins immobilized on plastic. At various time points, Jurkat cells were lysed and examined for c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) activity. RESULTS Soluble and immobilized GST-Tat2E, but not GST-Tat1E, GST-TatArg(mut) and GST-TatdeltaN, activated JNK in a dose-dependent manner, induced a rapid phosphorylation of c-Jun on Ser63 and promoted the de novo synthesis of c-Jun protein. Moreover, both GST-Tat2E and GST-Tat1E also stimulated ERK/MAPK. However, the activation of JNK was maximal at concentrations of 100 nM of GST-Tat2E and was blocked by the S6-kinase inhibitor rapamycin, whereas the activation of ERK/MAPK was already maximal at 1 nM of GST-Tat2E and was enhanced by rapamycin. CONCLUSIONS Tat-mediated activation of JNK requires the second exon of Tat, which is dispensable for the activation of ERK/MAPK. The ability to stimulate JNK and ERK/MAPK does not require Tat internalization.