Paola Comi

δβ-Thalassemia is due to a gene deletion

Abstract DNA has been prepared from peripheral blood or cultured skin fibroblasts obtained from three Sicilian and one Greek δβ-thalassemia homozygotes. Globin-gene analysis was carried out using a cDNA β probe, and the results indicate that δβ-thalassemia has arisen from a deletion of the β-globin genes. A similar result was obtained using DNA prepared from cultured skin fibroblasts from an individual homozygous for the Negro form of hereditary persistence of fetal hemoglobin (HPFH). In both cases, the deletion has spared the Gγ and Aγ loci directing the γ chains of hemoglobin F, but it has not been possible to demonstrate any difference between the size of the deletion involved in the production of δβ-thalassemia and that which gave rise to HPFH. These experiments provide further direct evidence that deletions of critical areas of the γ-δ-β gene cluster result in persistent γ chain synthesis in adult life.

Reciprocal regulation of Notch and PI3K/Akt signalling in T‐ALL cells In Vitro

Notch signalling plays an important role in hematopoiesis and in the pathogenesis of T‐ALL. Notch is known to interact with Ras and PTEN/PI3K (phosphoinositide‐3 kinase)/Akt pathways. We investigated the interaction of Notch with these pathways and the possible reciprocal regulation of these signalling systems in T‐ALL cells in vitro. Our analyses indicate that the PI3K/Akt pathway is constitutively active in the four T‐ALL cell lines tested. Akt phosphorylation was not altered by the sequestration of growth factors, that is, Akt activation seems to be less dependent on but not completely independent of growth factors, possibly being not subject to negative feedback regulation. PTEN expression was not detected in 3/4 cell lines tested, suggesting the loss of PTEN‐mediated Akt activation. Inhibition of the PI3K/Akt pathway arrests growth and enhances apoptosis, but with no modulation of expression of Bax‐α and Bcl‐2 proteins. We analysed the relationship between Notch‐1 and the PI3K/Akt signalling and show that inhibition of the Akt pathway changes Notch expression; Notch‐1 protein decreased in all the cell lines upon treatment with the inhibitor. Our studies strongly suggest that Notch signalling interacts with PI3K/Akt signalling and further that this occurs in the absence of PTEN expression. The consequences of this to the signalling outcome are yet unclear, but we have uncovered a significant inverse relationship between Notch and PI3K/Akt pathway, which leads us to postulate the operation of a reciprocal regulatory loop between Notch and Ras‐PI3K/Akt in the pathogenesis of T‐ALL. J. Cell. Biochem. 103: 1405–1412, 2008.

Globin gene deletion in HPFH, δ°β° thalassaemia and Hb Lepore disease

THE thalassaemias are a group of inherited disorders characterised by the defective production of either α (α thalassaemias) or non-α (β and δ°β° thalassaemias) globin chains of haemoglobins (Hb)1. In β thalassaemias the decreased synthesis of β-globin chains is only partially compensated by the increased production of γ chains, which probably reflects2 the massive hypertrophy of the erythron with selective survival of the clones of adult haemoglobin F-producing cells (F cells3,4). The situation is very different in other genetic disorders of the non-α gene cluster, known as δ°β° thalassaemias and Negro type of hereditary persistence of fetal haemoglobin (HPFH). In these two forms there is a genuine increase of γ-chain production, as shown by the high level of HbF found in heterozygotes. Although a clearcut distinction from both the clinical and haematological point of view cannot be traced between these two forms, the HPFH differs from the δ°β° thalassaemia in having a higher degree of γ-chain synthesis and a more homogeneous distribution of HbF within red cells. Recently, it has been possible to carry out gene analysis on DNA prepared from β°, δ°β° thalassaemic and HPFH patients. The β-globin gene is present in β° thalassaemias5–9, but in δ°β° thalassaemias and HPFH a major deletion, possibly involving both δ and β genes, has been demonstrated by hybridisation studies8,10–12. To characterise the molecular defect in these genetic disorders more precisely, we have hybridised DNA from homozygotes with HPFH, δ°β° thalassaemia and Hb Lepore disease (in which non-α-chains are a δβ fusion product13). For this we used a pure full-size cDNAβ probe and specific 5′ end and 3′ end cDNA fragments (we designate as 5′ end cDNA the portion corresponding to the 5′ end of the mRNA; the same for the 3′ end). Our results, reported here, show that in contrast to HPFH, where a complete δ and β gene deletion occurs, in δ°β° thalassaemia a 5′-end fragment of the δ gene is present.

Gγ and Aγ globin chains separation and quantitation by isoelectric focusing

Abstract Isoelectric focusing in the presence of Nonidet P-40 splits human chromatographically pure γ globin chains into two bands of isoelectric points 6.95 and 6.85, respectively. The comparison of the relative proportions of the two bands with the ratios between the Gγ and Aγ non allelic chains of human fetal hemoglobin suggests that the band at pI 6.95 corresponds to Gγ and the band at pI 6.85 corresponds to the Aγ chain; the latter is the only band present in a patient with Greek type hereditary persistence of fetal hemoglobin, producing only Aγ chains. Fluorography of electrofocusing-separated radioactive γ globin chains synthesized by thalassemic reticulocytes indicates that the relative G γ A γ synthetic ratios are similar to the relative amounts of Gγ and Aγ chains accumulated in the erythrocytes, suggesting that the activities for the Gγ and Aγ mRNAs decay at roughly similar rates.

Oxidative stress signalling in the apoptosis of Jurkat T lymphocytes

The pathways of transduction of oxidative stress signals have been studied using the Jurkat T cell model. The oxidative stress was induced by exposure of the cells to 100 μM H2O2. DNA damage was detected within 15 min after commencement of treatment. DNA damage repair occurred within about 1 h in cells exposed to oxidative stress for 15 min. In continuous exposure to stress, DNA repair was slower and control levels of DNA integrity were not reached. DNA repair did not involve gene transcription. H2O2 at 100 μM caused cell death by necrosis as well as by apoptosis. Both these processes were induced by 15 min exposure to the stress stimulus. However, some important differences were found between necrosis and apoptosis. Necrosis was more rapid, began within an hour of treatment and continued to increase during the full duration of the experiment. But apoptosis was seen after 4 h from treatment and was conspicuous between 6 and 20 h after the start of treatment. The necrotic phase preceded apoptosis, although these did show an overlap. In the necrotic phase, Bcl‐2, Caspase 8 genes were down regulated. The 6–20 h phase characterised by a marked increase in apoptosis is accompanied by the up regulation of both Bcl‐2 and Caspase genes. Expression of the Fas and p53 genes was not altered in either phase. We also analysed the levels of expression of the scavenging genes whose gene products are involved in detoxification. No modulation of the antioxidant enzymes, catalase, Cu/Zn superoxide dismutase and glutatione peroxidase was detectable. J. Cell. Biochem. 82:437–444, 2001.

A direct estimate of the number of human γ-globin genes

Abstract The number of genes specifying human γ-globin has been determined directly by hybridization of complementary DNA to total human DNA. The complementary DNA was enriched in sequences specific for γ-globin genes by transcribing globin mRNA isolated from fetal reticulocytes with viral reverse transcriptase, and collecting the material which does not back-hybridize to adult globin mRNA. When hybridized in cDNA excess to DNA, very similar values are found for γ-gene number as for β-gene number, suggesting two or at most three γ-globin genes per haploid human genome. This indicates that the non-Mendelian ratios of γ-chain mutants found in heterozygotes are due to transcriptional or post-transcriptional regulation rather than to gene dosage. The number of each major human globin gene has now been determined directly by molelcular methods.

OVER-EXPRESSION OF HEPATOCYTE GROWTH FACTOR IN HUMAN KAPOSI'S SARCOMA

Kaposis sarcoma is a highly vascularized multifocal tumor which frequently appears as a complication of HIV infection. It has been suggested that a disorder in the cytokine network may contribute to the development of the disease. We examined the expression of several cytokines in human sporadic Kaposis sarcoma specimens, as well as in spindle cells cultured from human lesions, and consistently found high levels of expression of hepatocyte growth factor (HGF). In addition, human lesion‐derived spindle cells synthesize and secrete biologically active hepatocyte growth factor and express the hepatocyte‐growth‐factor receptor (c‐MET). Moreover, elevated levels of transforming growth factor β1 (TGFβ1) mRNA were found in lesions of human sporadic Kaposis sarcoma and in lesion‐derived spindle cells which also over‐express urokinase. Since HGF, TGFβ1 and urokinase are all involved in capillary‐vessel organization, dysregulation of these interacting agents may play a role in the initiation and/or progression of Kaposis sarcoma, stimulating the growth of spindle cells and recruiting endothelial cells into the lesion.

Notch-ing from T-cell to B-cell lymphoid malignancies.

Notch receptors are transmembrane proteins critically determining cell fate and maintenance of progenitor cells in many developmental systems. Notch signaling is involved in stem cell self-renewal and regulates the main functions of cell life at different levels of development: cell proliferation, differentiation and apoptosis. By virtue of its involvement in the regulation of cell physiology, it is not surprising that a deregulation of the Notch pathway leads to the development of different tumors. In this review, we critically discuss the latest findings concerning Notch roles in hematologic oncology, with a special focus on T-cell acute lymphoblastic leukemia and B-cell malignancies. We also describe the molecular mediators of Notch-driven oncogenic effects and the current pharmacological approaches targeting Notch signaling.

Direct demonstration of beta-globin mRNA in homozygous Ferrara betaO-thalassaemia patients.

In cases of β0- thalassaemia from Ferrara, Italy, the β-globin gene is transcribed into mRNA but no protein is synthesised. For these cases there is no hybridisation data suggesting a globin gene structural mutation. This again demonstrates the diverse molecular events which may cause this prevalent hereditary disease.

Differential regulation of Notch signal transduction in leukaemia and lymphoma cells in culture.

The transduction of Notch signal plays an intricate role in cell differentiation and pathogenesis of haematological malignancies as well as in certain congenital conditions. We found no genomic changes in either gene in 34 leukaemic samples and 25 leukaemia and lymphoma cell lines. The functionality of Notch signalling was tested using HES1 gene activation. We show that Notch signalling is differentially regulated in T‐acute lymphoblastic leukaemia (ALL) and B‐lymphoma cells. The Notch pathway is intact in a majority of B‐lymphoma cell lines, but EBNA2, which mimics notch function, can occasionally activate the pathway. In contrast, the Notch pathway is constitutively active in T‐ALL. This is the first demonstration of a distinction between B‐lymphomas and T‐cell leukaemias in the functioning of the Notch‐signalling pathway. This might be related to their pathogenesis.