Antonio Fantoni

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.

The 5′ sequence of human Factor XII gene contains transcription regulatory elements typical of liver specific, estrogen-modulated genes

The human Factor XII gene codes for a serine proteinase synthesized in liver that activates both the coagulation and the fibrinolytic cascades. The nucleotide sequence analysis of a HincII-HincII 3129 bp fragment was performed showing that the FXII promoter region contains neither CAAT and TATA regulatory elements, nor GC islands, but revealing the presence of two tandemly repeated sequences in opposite orientation, two LF-A1 elements typical of the liver specific genes and one estrogen responsive element, that substantiates the observation of Factor XII gene modulation by estrogens.

Murine hepatocyte cell lines promote expansion and differentiation of NK cells from stem cell precursors

While fetal liver is a major hematopoietic organ, normal adult liver provides a suitable microenvironment for a variety of immune cells and, in several pathological conditions, may become a site of extramedullary hematopoiesis. The direct influence of hepatocytes on hematopoietic cell differentiation is poorly understood. We have previously reported that the Met murine hepatocyte (MMH) untransformed hepatocytic lines retain several morphological and functional features of hepatocytes in vivo and are able to support the survival, self‐renewal, and differentiation of hematopoietic precursors in a cell‐cell contact system. Here we report the effects of soluble factors released by MMH lines on bone marrow–derived cells. Lymphohematopoietic cells were cultured in two different cell contact‐free systems: transwell inserts on MMH feeder layers, and MMH conditioned medium (MMH‐CM). Both culture systems were able to promote a substantial expansion of bone marrow‐derived cells and their differentiation to natural killer (NK) cells that express the NK1.1 and U5A2‐13 markers. Purified hematopoietic stem cells (Sca‐1+Lin‐), either plated as a bulk population or as single cells, were also able to differentiate into NK cells, when cultured in MMH‐CM; thus, soluble factors secreted by MMH lines promote the expansion and differentiation of NK precursor cells. MMH‐CM‐derived NK cells are functionally active; stimulation by interleukin (IL)‐12 together with IL‐18 was required to induce interferon‐gamma (IFNγ) expression and to enhance their cytotoxic activity. In conclusion, our findings may imply a direct role of hepatocytes in NK cell development, and the system we have used may provide a tool for studying the molecular mechanisms of NK cell differentiation. (HEPATOLOGY 2004;39:1508–1516.)

Human CD26 expression in transgenic mice affects murine T-cell populations and modifies their subset distribution

CD26 is a type II transmembrane glycoprotein with dipeptidyl peptidase (DPPIV) activity, constitutively expressed in different cell types and contributing to T-cell activation by acting as costimulatory molecule. Although data suggest an important role for CD26 within the immune system, the physiologic function of this molecule is still unknown. To investigate the role of CD26 in vivo we have produced transgenic mice expressing the human molecule in T cells. Human CD26 (huCD26) is constitutively expressed in all thymocytes and peripheral T lymphocytes of these transgenic mice and is endowed with an enhanced DPPIV activity. CD26 transgene expression induces major phenotypic changes to T-cell populations within the thymus and in peripheral blood. After the onset of sexual maturity, huCD26 expression induces an age-related overreduction of thymus cellularity accompanied by a relative impairment of thymocyte proliferation following lectin stimulation. Also the peripheral blood T-cell pool is reduced in huCD26 transgenic mice and this is accompanied by an increase of the apoptotic rate of CD4+ and CD8+ subpopulations. Taken together these data suggest that CD26 interferes with transduction pathway(s) needed for the maturation of T cells and plays an important role in T lymphocyte homeostasis in peripheral blood.

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.

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.

Membrane expression of HLA-Cw4 free chains in activated T cells of transgenic mice

Transgenic nice were produced in which human HLA-Cw4 is stably integrated, behaves as a single Mendelian trait, and, being under the transcriptional control of humanCD2, is selectively and efficiently expressed in T lymphocytes. These mice were used as a model system to dtermine whether HLA-type C molecules can be exposed on the surface of activated lymphocytes as free heavy chains, non-associated with β2-microglobulin (β2m). In our transgenic mice we could identify HLA-Cw4 molecules either as free chains or as β2m-associated molecules by the use of monoclonal antibodies specific for either conformation of HLA class I and nonreactive to mouse H2 molecules. Resting mouse lymphocytes were shown by western transfer analysis to contain sizeable amounts of HLA-Cw4 free chains, but they exposed on thier surface HLA-Cw4 only in association with β2m,a s indicated by flow cytometric measurements. Conversely, where the content of total HLA-Cw4 was increased, lectin-activated mouse lymphocytes exposed on thier outer cell membrane HLA-Cw4 molecules in both conformation, namely, also as free heavy chains, Isoelectrofocusing analysis confirmed the presence of both HLA-Cw4 molecular conformations in activated T cells and indicated that HLA-Cw4 heavy chains can bind to mouse β2m with the same low affinity displayed for human β2m. The results of our experiments led us to conclude that (1) association with β2m is not necessary for the exposure of HLA-C on the surface of activated T lymphocytes and (2)o cell activation affects the balance between the two conformation forms of HLA-C.

Assignment of human coagulation factor XII (fXII) to chromosome 5 by cDNA hybridization to DNA from somatic cell hybrids

SummaryHuman coagulation factor XII (fXII), a serine protease synthesized in liver and active in plasma, is involved in a wide variety of functions, including blood coagulation, fibrinolysis, bradykinin and complement activation. A complementary DNA (597 bp) encoding amino acid-16 to amino acid 183 of fXII protein was used to determine the chromosomal location of the fXII gene. DNAs from hamster-human somatic cell hybrids were digested with restriction enzymes and hybridized with the fXII cDNA. By the Southern method it was shown that restriction fragments able to hybridize to fXII cDNA are present only in DNA extracted from clones retaining human chromosome 5.

Human CD4 produced in lymphoid cells of transgenic mice binds HIV gp120 and modifies the subsets of mouse T-cell populations.

Studies of the interaction between human and mouse molecules endowed with T-cell functions may add important information to our understanding of the functional organization of these molecules, mainly T-cell receptor (Tcr), major histocompatibility complex (MHC), the CD4 and CD8 coreceptors, and p56 lck. The expression of the human CD4 gene in transgenic mice may represent a useful tool for the exploration of the relationship of CD4 molecules with different ligands and receptors involved in T-cell functions. The compatibility of species-specific structures with the overall CD4 function was previously studied by transfection experiments (Turner et al. 1990; Collins et al. 1992). These studies have indicated that the human CD4 (HuCD4) gene expressed in cultured mouse cells can associate with mouse p56 lck and, in addition (yon Hoegen et al. 1989; Glaichenhans et al. 1991), that it can interact functionally with mouse class II MHC molecules. More conclusive evidence concerning this issue can be drawn from in vivo studies: namely, of transgenic mice expressing the human CD4 gene. Barzaga-Gilbert and co-workers (1992) have reported an authentic interaction between HuCD4 exposed on T cells from transgenic mice and class II MHC molecules from human cells. We are interested in exploring the possible function of HuCD4 in the distribution of mouse T-cell populations both in thymus and in lymphoid peripheral organs of HuCD4 transgenic mice. For such in vivo studies it is critical to show that HuCD4 is exposed on mouse CD4 ÷ T-cell membranes at a per cell density equal to that of human CD4÷ T-cell membranes, and also to demonstrate that the correct conformation and trans-

α1-antitripsina umana: diagnosi molecolare di mutazione e modelli animali di patologia umana

Un gene mutato per ľα1-antitripsina (α1AT) umana e stato isolato ed analizzato a livello molecolare. La mutazione induce bassi livelli plasmatici della proteina ed una importante patologia polmonare ed epatica. Ľallele mutato considerato (allele Z) presenta due mutazioni puntiformi: una e a livello del V esune del gene e si ritrova in tutti i soggetti carenti, ľaltra e nel III esone ed e presente sia nei soggetti carenti che nel 23% dei soggetti normali. Per identificare sui genoma totale il gene delľα1AT abbiamo sintetizzato quattro oligonucleotidi, complementari a sequenze fiancheggianti le due mutazioni, che funzionano da primer per una particolare DNA polimerasi termoresistente. Il III esone amplificato e stato digerito con ľenzima di restrizione BstEII il cui sito, normalmente presente, viene soppresso dalla mutazione. I due esoni amplificati sono stati quindi sequenziari e inseriti separatamente in un vettore plasmidico. I nostri risultati permettono: a) la diagnosi molecolare di mutazione e la diagnosi prenatale dei feti a rischio; b) la costruzione di geni anomali utilizzabili nella costruzione di animali transgenici.A mutant gene for α1-antitrypsin (αlAT) was isolated and analyzed to determine a method for molecular diagnosis of the protein deficiency, which is involved in severe liver and lung diseases. The mutant gene (called Z allele) has two point mutations: the first mutation is located in exon V and is present in all the subjects with protein deficiency; the other one is located in exon III and is present in the same subjects and in 23% ot normal individuals. To identity exon III and exon V ot α1AT among the total genome we synthesized lour oligonucleotides that are complementary to sequences flanking the mutation sites and act as primers for a polymerization chain reaction. The amplified fragments were digested with BstEII enzyme. The restriction site is present in exon III ot the normal gene and is abolished by the mutation in exon III of the Z gene. The amplified DNA fragments were cloned and sequenced. These results allow: a) a molecular diagnosis of the mutation (even in at risk fetuses); b) the construction of mutant genes tor production or transgenic mice.RiassuntoUn gene mutato per ľα1-antitripsina (α1AT) umana è stato isolato ed analizzato a livello molecolare. La mutazione induce bassi livelli plasmatici della proteina ed una importante patologia polmonare ed epatica. Ľallele mutato considerato (allele Z) presenta due mutazioni puntiformi: una è a livello del V esune del gene e si ritrova in tutti i soggetti carenti, ľaltra è nel III esone ed è presente sia nei soggetti carenti che nel 23% dei soggetti normali. Per identificare sui genoma totale il gene delľα1AT abbiamo sintetizzato quattro oligonucleotidi, complementari a sequenze fiancheggianti le due mutazioni, che funzionano da primer per una particolare DNA polimerasi termoresistente. Il III esone amplificato è stato digerito con ľenzima di restrizione BstEII il cui sito, normalmente presente, viene soppresso dalla mutazione. I due esoni amplificati sono stati quindi sequenziari e inseriti separatamente in un vettore plasmidico. I nostri risultati permettono: a) la diagnosi molecolare di mutazione e la diagnosi prenatale dei feti a rischio; b) la costruzione di geni anomali utilizzabili nella costruzione di animali transgenici.