Biomedical subjects

M Ponzoni

Publications and source records attributed to M Ponzoni.

88 records · Page 5Linked to original sources

PubMed1989

Retinoic acid inhibits phosphatidylinositol turnover only in RA-sensitive while not in RA-resistant human neuroblastoma cells.

Phosphatidylinositol (PI) turnover has recently been implicated in the regulation of cell proliferation and transformation. We have investigated its role in differentiation using LAN-1 cells, a human neuroblastoma cell line which can be induced to differentiate along the neuronal pathway by retinoic acid (RA), and a derivated RA-resistant subline of it (LAN-1-res). We have found that treatment of LAN-1 cells with RA is followed by a rapid decrease of inositol phospholipid metabolism, using myo-[1,2-3H] inositol or [1,(3)-3H] glycerol. Analysis of labelled phosphatidylinositol metabolites from prelabelled LAN-1 cells indicated a rapid decrease of inositol (1,4,5)-trisphosphate and (1,2) diacylglycerol within 1 min. of induction of differentiation by RA, while no changes were observed in RA-treated LAN-1-res cells. These findings indicate that phosphoinositides-derived metabolites may be directly implicated in the induction processes of RA-triggered NB cell differentiation.

Cell Differentiation↗

PubMed1989

Phosphatidylinositol turnover is not a general regulator of neuroblastoma cell differentiation: comparison between two differentiating agents, retinoic acid and gamma-interferon.

The turnover of phosphatidylinositol (PI) is believed to constitute a crucial step in the signaling pathways for stimulation of cells by a variety of bioactive substances, including differentiating agents; however decisive evidence for the idea has not been obtained. In the present paper, we investigated the involvement of PI turnover in cell differentiation using a human neuroblastoma cell line, LAN-1, which can be induced to differentiate along the neuronal pathway by both retinoic acid (RA) and gamma-interferon (gamma-IFN). Analysis of labelled phosphatidylinositol metabolites from prelabelled cells indicated a rapid decrease of inositol 1,4,5-trisphosphate and 1,2-diacylglycerol within 1 min of induction of LAN-1 cell differentiation by RA, while no changes were observed in gamma-IFN-treated cells. These findings indicate the occurrence of decreased inositol phospholipid turnover in RA-treated LAN-1 cells and suggest that phosphoinositide-derived metabolites may not constitute general regulators of cellular differentiation.

Cell Differentiation↗

PubMed1989

Morphologic and phenotypic changes of human neuroblastoma cells in culture induced by cytosine arabinoside.

The effects of cytosine-arabinoside (ARA-C) on the growth and phenotypic expression of a new human neuroblastoma (NB) cell line (GI-ME-N) have been extensively tested. Low doses of ARA-C allowing more than 90% cell viability induce morphological differentiation and growth inhibition. Differentiated cells were larger and flattened with elongated dendritic processes; such cells appeared within 48 h after a dose of ARA-C as low as 0.1 micrograms/ml (about 1000-fold lower than the conventional clinic dose). The new morphological aspect reached the maximum expression after 5-6 days of culture being independent from the addition of extra drug to the culture. A decrease in [3H]thymidine incorporation was also observed within 24 h and the cell growth was completely inhibited on the sixth day. Moreover, ARA-C strongly inhibited anchorage-independent growth in soft agar assay. Membrane immunofluorescence showed several dramatic changes in NB-specific antigen expression after 5 days of treatment with ARA-C. At the same time ARA-C also modulated cytoskeletal proteins and slightly increased catecholamine expression. These findings suggest that noncytotoxic doses of ARA-C do promote the differentiation of GI-ME-N neuroblastoma cells associated with reduced expression of the malignant phenotype.

Cell Differentiation↗

PubMed1989

Effects of gamma-interferon on the growth, morphology, and membrane and cytoskeletal proteins expression of Lan-1 cells.

The effects of gamma-interferon (gamma-IFN) on the growth, morphology, and phenotypic expression of the human neuroblastoma (NB) cell line, LAN-1, have been extensively tested. Low doses of gamma-IFN allowing more than 90% cell viability induce morphological differentiation and growth inhibition. Cells exposed to gamma-IFN significantly decreased their growth rate, became smaller and poligonal, and sprouted long cellular processes with varicosities along their course, typical of the neurites seen in differentiated NB cells; morphological changes appeared within 48 h of culture with 1,000 U/ml gamma-IFN. The new morphological aspect reached the maximum expression after 6 days of culture, becoming more evident when fresh drug was added after 2 days of culture. A decrease in [3H]thymidine incorporation was also observed within 24 h; cell growth was completely inhibited at the 6th day. Membrane immunofluorescence showed several changes in NB-specific antigen expression after 6 days of treatment with gamma-IFN. At the same time gamma-IFN also modulated cytoskeletal proteins. These findings suggest that noncytotoxic doses of gamma-IFN do promote the differentiation of LAN-1 neuroblastoma cells which is associated with the reduced expression of the malignant phenotype.

Cell Differentiation↗

PubMed1988

Purification to homogeneity and biochemical characterization of two suppressor factors from human malignant T-cells.

A cell clone (GI-CO-T-9) derived from a long term T-cell culture (PF-382), established from a patient affected by acute T-lymphoblastic leukemia (T-ALL), was selected for the presence in the culture medium of factors suppressing T-cell proliferation. The crude supernatant has been subjected to a multi-step chromatographic fractioning, including: preparative gel permeation, anion exchange, and hydrophobic interaction High Performance Liquid Chromatography (HPLC). The highly purified material was characterized by polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE), revealing single bands of 115 Kd and 80 Kd. The isoelectric points (pI), determined by flat-bed isoelectric-focusing, were 7.4 for High Molecular Weight Suppressor Factor (HMWSF) and 3.5-3.6 for Low Molecular Weight Suppressor Factor (LMWSF).

Cell Line↗

PubMed1988

A human acute leukemia-derived T-cell line produces two inhibitor factors which suppress lymphocyte proliferation: characterization and purification of the molecules.

The crude supernatant of an CD1+/CD8+ T-cell clone (GI-CO-T-9) established from a long standing culture of an acute T-lymphoblastic leukemia (T-ALL) was shown to inhibit the responsiveness of normal PBMC to PHA. This clone does not produce TNF-alpha, TNF-beta, alpha-IFN, tau-IFN, IL-1, IL-2 and has no NK-like activity. The crude supernatant has been subjected to a multi-step chromatographic fractioning. Preparative gel permeation HPLC allowed us to recover two peaks of biologic activity in the range of 100-120 kDa and 75-85 kDa (referred to as "high molecular mass inhibitor factor", HMMIF, and "low molecular mass inhibitor factor", LMMIF, respectively). Both fractions were then subjected to anion exchange HPLC: HMMIF was recovered in fractions eluting at 0.04 M NaCl while LMMIF eluted at higher ionic strength (0.48 M NaCl). The fractions with biologic activity recovered from anion exchange HPLC have been subjected to hydrophobic interaction HPLC (HIC) for final purification. The highly purified material was characterized by polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate (SDS) revealing single bands of 115 kDa and 80 kDa. The isoelectric points (pI), determined by flat-bed isoelectricfocusing, were 7.4 for HMMIF and 3.5-3.6 for LMMIF. Studies on temperature lability indicated that both proteins are stable for 3-4 hours at room temperature (RT), 24-36 hours at +4 degrees C and 7-10 days at -80 degrees C.

Chromatography, High Pressure Liquid↗

PubMed1988

[Immunosuppressive factors produced by a T cell line derived from acute lymphoblastic leukemia].

The supernatant of CD8+ cells, isolated from a permanent lymphoblastoid cell clone established from a long term culture of a T cell acute lymphoblastic leukemia, contained two distinct molecules with suppressive activity on PHA1-induced PBMC proliferation. This clone does not produce TNF-alpha, TNF-beta, alpha-IFN, gamma-IFN, IL-1, IL-2 and has not natural killer activity. In the attempt to purify and biochemically characterize the lymphoblastic cell line-derived T-cell SFs, a multi-step chromatographic separation has been used. Two different peaks of biologic activity have been separated by HPLC gel permeation in the range of 100-120 Kd and 75-85 Kd referred to as high molecular weight suppressor factor HMWSF, and low molecular weight suppressor factor LMWSF, respectively. These fractions were then concentrated, dialyzed and further purified by anion exchange HPLC. This chromatographic step allowed us to considerably purify the two SFs. The biologically active fractions derived from the previous chromatographic step were eventually subjected to hydrophobic interaction HPLC (HIC) for final purification. The highly purified material was characterized by polyacrylamide gel electrophoresis in sodium-dodecylsulfate (SDS-PAGE): a single band corresponding to 115 Kd was observed for HMWSF, while LMWSF yielded a single band at 80 Kd. The isoelectric points (pI) of the different SFs was determined by flat-bed isoelectric-focusing: the HMWSF yielded a single band at pI 7.4, while a much lower pI was observed for LMWSF, 3.5-3.6. Studies on temperature lability indicated that both proteins are stable for 3-4 hours at room temperature (RT), 24-36 hours at +4 degrees C and 7-10 days at -80 degrees C.

Antigens, CD↗

PubMed1988

[A rapid decrease in the phosphatidylinositol cycle during neuroblastoma cell differentiation induced by retinoic acid].

Inositol phospholipid turnover is part of a signal transduction mechanism which mobilize intracellular calcium and activate a calcium- and phospholipid-dependent protein kinase, protein kinase C. Phosphatidylinositol turnover has recently been implicated in the regulation of cell proliferation and transformation. Its role in differentiation has now been investigated using LAN-1 cells, a human neuroblastoma cell line which can be induced to differentiate along the neuronal pathway by RA. Treatment of LAN-1 cells with RA was followed by a rapid decrease of inositol phospholipid metabolism, as determined by isotopic methodology employing myo-[1,2-3H] inositol or [1(3)-3H] glycerol. Analysis of labelled phosphatidylinositol metabolites from prelabelled cells indicated a rapid decrease of inositol (1,4,5)trisphosphate and (1,2)diacylglycerol within 1 min. of induction of LAN-1 cell differentiation. These findings suggest that inositol phospholipid-derived metabolites (i.e. diacylglycerol and inositol trisphosphate) may be part of the mechanism by which certain RA signals are transduced, playing a key role in control of neuroblastoma cell differentiation.

Cell Differentiation↗

PubMed1988

Effect of cytosine arabinoside on the growth and phenotypic expression of GI-ME-N, a new human neuroblastoma cell line.

Cytosine-arabinoside (ARA-C) effects on a new human neuroblastoma cell line (GI-ME-N), recently established in our laboratory, have been extensively tested. Low doses of ARA-C allowing virtually 100% cell viability induce morphological differentiation and growth inhibition; differentiated cells appear larger and flattened with elongated dendritic processes; such cells appeared within 48 hours after a dose of ARA-C as low as 0.1 microgram/ml. The new morphological aspect reached the maximum expression after 5-6 days of culture being independent of the addition of extra drug to the culture. A decrease in (3H)-thymidine incorporation was also observed within 48 hours being the cell growth completely inhibited at the 6th day. Membrane immunofluorescence with specific monoclonal antibodies showed several dramatic changes in NB-specific antigen expression after 5 days of treatment with ARA-C. These findings suggest that low ARA-C doses promote the differentiation of GI-ME-N neuroblastoma cells resulting in an altered expression of the malignant phenotype.

Cell Division↗

PubMed1987

Interleukin 2 rapidly stimulates synthesis and breakdown of polyphosphoinositides in interleukin 2-dependent, murine T-cell lines.

Several T-cell functions are controlled by the regulatory peptide interleukin 2 (IL-2). Binding of IL-2 with specific receptors has been well documented, but the molecular mechanism by which IL-2/IL-2 receptor interaction is transduced is not known. We have found that treatment of IL-2-dependent T-cell lines with IL-2 is followed by a rapid stimulation of inositol phospholipid metabolism, as determined by isotopic methodology employing myo-[1,2-3H]inositol. Increased incorporation of the metabolic precursor into phosphatidylinositol and phosphatidylinositol 4-monophosphate, together with the appearance of radiolabeled phosphatidylinositol 4,5-bisphosphate, occurred within minutes of treatment with IL-2 of factor-dependent CT6 cells. Analysis of labeled water-soluble compounds from prelabeled cells indicated a rapid (within 1 min) stimulation of inositol phospholipid hydrolysis following IL-2 treatment. Increased recovery of [3H] inositol phosphates and appearance of [3H]inositol trisphosphate were observed after treatment with IL-2 of CT6 cells, as well as of a second IL-2-dependent cell line, CTB6. These findings suggests that inositol phospholipid-derived metabolites (i.e. diacylglycerol and inositol trisphosphate) may be part of the mechanism by which certain IL-2 signals are transduced.

Animals↗

PubMed1985

[In vitro synthesis of interleukin 2 (IL-2) by T lymphocytes extracted from a lymphoepithelial thymoma. Purification and biochemical characterization].

T (E+) lymphocytes purified from lymphoepithelial thymoma surgically removed from an asymptomatic 12 yrs old girl were cultured in presence of PHA-P; culture supernatant contained a T cell growth factor (IL-2) biochemically different from that purifiable from supernatants of normal T cells. Differences in molecular weight, isoelectric point and ion exchange elution pattern would allow to compare this abnormal IL-2, (named Thy-IL-2 from Thymus) to that already characterized from the HUT 102 T cell line (L-TCGF peak II). A comparison of biochemical properties of Thy-IL-2 and normal IL-2 (N-IL-2) is also presented; the biological significance of such differences are discussed.

Child↗

PubMed1985

T cells from lymphoepithelial thymoma produce a biochemical variant of IL-2.

The supernatant of TE+ lymphocytes isolated by density gradient from a lympho-epithelial thymoma contained a biochemical variant of T cell growth factor (IL-2). This protein showed two peaks with an apparent molecular weight of 18 and 28 kd by Sephadex G 100 chromatography. Further purification was achieved by DEAE Sephacel and CM Sepharose ion-exchange chromatography. By isoelectric focusing the isoelectric point was found to differ from that of normal. IL-2. Based on these results, the IL-2 variant appears to be a new kind of lymphokine distinguishable from normal IL-2 and similar to a TCGF (L-TCGF peak II) purified from the malignant T cell clone HUT 102.

Chromatography, Gel↗

PubMed1982

Immune evaluation of 50 children with neuroblastoma at onset.

Several studies document the importance of immunity in the host-tumor relationship in neuroblastoma patients, evidentiating a correlation between clinical compromission and immune impairment. In this study we evaluated some aspects of cellular and humoral immune capacity in 50 neuroblastoma patients at onset. The aim of this investigation was to define, if any, the common immune pattern of these patients, and to evidentiate a possible correlation with prognosis. Immune tests performed were serum immunoglobulin quantitation, absolute value of total T and B lymphocytes, and lymphocyte reactivity to phytohemagglutinin (PHA), Concanavalin A (ConA), and Pokeweed mitogen (PWM). In patients with localized or regional disease, diminished values of lymphocytes were observed in 4/16 cases, a datum highly correlated to a poor final outcome (P less than 0.01). Mitogen response and serum immunoglobulin levels were frequently altered, but no prognostic value was evidentiated. Thirty-one children with disseminated disease presented more frequent and extended abnormalities: No parameter was found to correlate with prognosis, except for an impaired PHA response that paradoxically assumed a favourable prognostic meaning. Our results suggest that only the total lymphocyte number can contribute to predict the survival ratio in patients with regional disease.

Adolescent↗

PubMedDate not supplied

In vitro pharmacology of metaiodobenzylguanidine uptake, storage and release in human neuroblastoma cells.

Four human neuroblastoma (NB) cell lines (LAN-5, SK-N-BE(2)C, GI-LI-N, and GI-CA-N) have been investigated for their ability to take up and store [125I]metaiodobenzylguanidine (125I-MIBG) in vitro. Only SK-N-BE(2)C and LAN-5 cells were able to specifically take up MIBG, with the former cell line showing a more efficient retention of the radiotracer. 125I-MIBG incorporation in both cell lines was inhibited by norepinephrine, desipramine, ouabain and energy depletion. Thus, all the major criteria for specific (type 1) uptake were fulfilled. Conversely, GI-LI-N and GI-CA-N cells did not show any specific uptake. Pharmacological manipulations aimed at defining the intracellular site(s) of 125I-MIBG storage clearly showed that the radiotracer is not accumulated in the reserpine-sensitive neurosecretory granules and vesicles in NB cells, contrary to what has been observed in a chromaffin derived tumor cell line (PC12). Our study provides new and suitable models to investigate in vitro the molecular and cellular pharmacology of MIBG in NB cells.

3-Iodobenzylguanidine↗

PubMedDate not supplied

[Neuroblastoma as a model for the in-vitro study of differentiated therapy].

Differentiation of human neuroblastoma (NB) cells is a very interesting biologic event, providing useful insights in both basic neurobiology and clinical management of this malignancy. Investigation of in vitro NB differentiation exploits several NB cell lines that can be induced to differentiate by an array of natural or synthetic chemicals, as well as biological factors such as some cytokines. The hallmarks of neuronal differentiation are represented by a partial or complete block of cell proliferation, morphological alterations and acquisition of biological features typical of mature neurons (for example, induced synthesis and storage of monoamines and neuropeptides, expression of peculiar cytoskeletal proteins and membrane antigens). The possibility to transfer the differentiative approach to the treatment of NB patients opens exciting therapeutic perspectives.

Cell Transformation, Neoplastic↗

PubMedDate not supplied

[Clinical value of spleen autograft].

A relaparotomy for cholecystectomy in a 21 year old patient, who received a spleen autograft after a traumatic injury four years before, allowed us to describe the long term morphological and functional follow-up of the autotransplanted spleen, with a histological evaluation of one of the grafts. We point out the technical aspects of the surgical procedure with a review of the experimental background. We underline the analogy of the functional features of the splenic autograft both in the experimental field and in the clinical setting. The histological study of the splenic fragment in our case showed the same structural pattern of the experimental studies. Thus we stress the hypothesis of an effective protection of the implanted splenic tissue against the overwhelming post-splenectomy sepsis in man.

Adult↗