Roberto Gambari

Human leukemia K-562 cells: induction of erythroid differentiation by 5-azacytidine.

In this article we show that the cytidine analog 5-azacytidine is able to induce differentiation of the human leukemia K-562 cell line. Erythroid induction is associated with (a) an increase of the overall globin synthesis and globin mRNA accumulation, (b) a relative increase of fetal with respect to embryonic globins, and (c) a decrease of the proliferative capacity of hemoglobin-containing cells. In addition, we have analysed the DNA methylation pattern at the cleavage sites of MspI and HpaII restriction enzymes, which are known to cleave differently CCGG DNA sequences when 5-methylcytosine is present. These experiments indicate that in K-562 cells treated with 5-azacytidine, DNA becomes hypomethylated, suggesting that genetic programmes leading to an erythroid phenotype may be activated by a reduction of DNA methylation.

Human leukemia K562 cells: relationship between hemin-mediated erythroid induction, cell proliferation and expression of c-abl and c-myc oncogenes.

We have studied the expression of the c-myc and c-abl oncogenes in two human leukemic K562 cell lines which do express hemoglobin genes retaining a differential rate of cell proliferation. Our data indicate that in hemin-induced K562(S) cells the expression of c-abl oncogene decreases and appears to be related to a decrease in the proliferation capacity rather than to the activation of differentiated functions. The K562(hC) cell line, which produces large amounts of Hb Gower 1 retaining an efficient rate of cell proliferation, expresses indeed the c-abl oncogene at high level.

Inhibition of immunodeficiency type-1 virus (HIV-1) life cycle by medicinal plant extracts and plant-derived compounds

Abstract Identification of molecules inhibiting the different steps of the life cycle of the human immunodeficiency virus type 1 (HIV-1) is central for the development of an efficient therapeutic treatment of AIDS. In this respect, this research takes great advantage from the fact that the molecular biology of the HIV-1 life cycle is well known. The present review summarizes and discusses strong evidences demonstrating that several medicinal plant extracts display anti-HIV-1 activities. Moreover, several compounds isolated from bioactive extracts were characterized in detail and found to exhibit inhibitory effects against different steps of the HIV-1 life cycle, including virus–cell fusion and virus absorption, reverse transcription, integration and proteolytic cleavage. Clinical trials on limited numbers of patients suffering from AIDS have been already reported, supporting the concept that some medicinal plant extracts or single molecules derived from them display anti-HIV effects not only in vitro but also in vivo . Therefore, medicinal plant extracts should be considered as an excellent source of clinically relevant anti-HIV-1xa0molecules.

Desferrioxamine inhibits induced erythroid differentiation of human leukemic K-562 cells

The iron chelator desferrioxamine reversibly inhibits heme accumulation and globin synthesis in human leukemic K-562 cells induced to express erythroid genes by butyric acid. These results suggest that iron metabolism can modulate globin gene expression. In addition we describe experimental conditions (6.25 micrograms/ml desferrioxamine) which do not suppress transcription of globin genes and translation of globin mRNA but prevent heme synthesis. Therefore expression of globin genes in butyric acid induced K-562 cells does not require accumulation of heme molecules. Human leukemic K-562 cells cultured with different inducers and treated with desferrioxamine should be used as a useful model system to further analyse the relationship between expression of erythroid genes, iron metabolism and heme accumulation.

Plants with antitumor properties: from biologically active molecules to drugs

Abstract Medicinal plants are of great interest as starting material for identification of new biologically active compounds. A large number of low-molecular-weight compounds isolated from plants or microorganisms have already been identified as effective in diseases as diverse as HIV infection, herpes simplex, neuroblastoma, and breast cancer. Some drugs that emerged from this process are already in the late-phase clinical trials. The first step for the identification of bioactive compounds in the biomedical field is the screening of extracts from different tissues of several medicinal plants for a given activity (for instance, in vitro antiproliferative activity). To this aim, the method of extraction of bioactive compounds is crucial. The second step is the fractionation and the characterization of the plant extracts exhibiting the desired biological activity. This step takes advantage from several analytical and preparative procedures, among which preparative and analytic high-performance liquid chromatography (HPLC), several HPLC-based methods, such as HPLC/MS, gas chromatography/mass spectrometry (GC/MS), surface plasmon resonance (SPR)-based biospecific interaction analysis (BIA) employing biosensors. Applications of these methodologies to the screening, identification, purification, and characterization of bioactive compounds from medicinal plants are described in this review.

Effects of plant extracts on gene expression profiling: from macroarrays to microarray technology

Abstract DNA hybridization arrays (macro- and microarrays) are very useful tools for the analysis of gene expression profiles in human pathologies. In addition, macro- and microarrays can be used in pharmacogenomic and toxicogenomic experiments, aimed at extensive analyses of the effects of therapeutic drugs on overall gene expression of target cells. Despite the fact that extracts from medicinal plants have been described to retain interesting biological activity, including anti-inflammatory, antitumor and antimicrobial effects, few data are present in the literature on gene expression profile studies. Here, we review results on the effects of plant extracts from Moringa oleifera on the gene expression profile of a human tumor cell line, the K562 cell line, originally isolated from a patient with chronic myelogenous leukemia (CML) in blast crisis, very useful for the identification of antitumor compounds. The data obtained using macroarrays were compared with those obtained using reverse-transcription polymerase-chain reaction (RT-PCR). Effects of M. oleifera extracts were compared to those of Emblica officinalis . The results obtained suggest that a general strategy for the development of specific therapeutic approaches could be proposed starting from gene expression studies employing macro- or microarrays. Treatment of target cells with plant extracts will allow to identify genes, which are down- or up-regulated. For these genes, the molecular analysis of the promoter regions and coding sequences could allow to design decoy oligonucleiodes (ODN), antisense DNA or RNA, peptides and monoclonal antibodies expected to mimic the biological effects of the employed plant extracts.

Therapy for Cystic Fibrosis Caused by Nonsense Mutations

Nonsense mutations cover about 10% of cystic fibrosis (CF) patients and generate premature termination codons (PTCs) leading to premature translational termination and causing the synthesis of truncated non-functional or partially functional CFTR (cystic fibrosis transmembrane conductance regulator) protein. The read-through approach is the suppression of translation terminations at PTCs and it has been developed as a therapeutic strategy to restore full-length protein using aminoglyco‐ side antibiotics or PTC124. Phenotypic consequences of PTCs can be exacerbated by the nonsense-mediated mRNA decay (NMD) pathway, which detects and degrades mRNA containing PTC. Therefore, modulation of NMD is also of interest as a potential target for suppression therapy. Not all PTCs are susceptible to the read-through treatment alone, especially where the nonsense mutations are combined with other CFTR mutations. For example, many CF patients present the highly frequent F508del CF mutation, causing an alteration of the cell membrane positioning of the CFTR channel. Pharmacological correctors that rescue the trafficking of F508del CFTR may overcome this defect. A combined administration of correctors/potentiators, readthrough molecules, and/or NMD inhibitors, depending on the genotype of the CF patients, could be the basis for the design of a personalized therapeutic approach.

Molecular cloning and sequence analysis of a cDNA coding for the mouse alpha-like embryonic globin chain x

Cytoplasmic poly(A)+mRNA from 12-day mouse-yolk-sac erythroid cells has been used to prepare a cDNA library in the plasmid pBR322. One clone containing sequences coding for the alpha-like embryonic globin chain x, pHE52, has been identified by hybrid selection and in vitro translation of the complementary mRNA. The nucleotide sequence of pHE52 confirms that it codes for an embryonic alpha-like globin chain. The insert sequence is 316 nucleotides long, contains the codons corresponding to amino acid residues 43-141, and extends into the 3 untranslated region. An analysis of the nucleotide sequence of pHE52 and the other known alpha globins suggests that the adult-embryonic divergence began approx. 400 million years ago reflecting a difference in the evolutionary history of the alpha- and beta-globin gene complexes.

Genetic Analyses in Health Laboratories: Current Status and Expectations

Genetic analyses performed in health laboratories involve adult patients, newborns, embryos/fetuses, pre-implanted pre-embryos, pre-fertilized oocytes and should meet the major medical needs of hospitals and pharmaceutical companies. Recent data support the concept that, in addition to diagnosis and prognosis, genetic analyses might lead to development of personalized therapy. Novel frontiers in genetic testing involve the development of single cell analyses and non-invasive assays, including those able to predict outcome of cancer pathologies by looking at circulating tumor cells, DNA, mRNA and microRNAs. In this respect, PCR-free diagnostics appears to be one of the most interesting and appealing approaches.