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Biomedical subjects

L Magnelli

Publications and source records attributed to L Magnelli.

At least 37 records · Page 2Linked to original sources
PubMed1995

Phorbol esters attenuate the expression of p53 in cells treated with doxorubicin and protect TS-P53/K562 from apoptosis.

The induction of p53 by doxorubicin in normal human fibroblasts was completely reverted by TPA, a phorbol ester. A ts-p53 mutant protein which is ineffective at 37 degrees C, but behaves in a wild-type fashion at 32 degrees C, was overexpressed in the p53-null human leukemia cell line K562. Wild-type-p53 overexpression induced apoptosis, whereas TPA protected K562 cells from this phenomena. By analogy with the observed human fibroblasts, TPA was found to decrease p53 amount. The TPA-dependent down-regulation of p53 could explain the chromosomal gross alterations typical of cells subjected to a chronic TPA treatment, alterations also found in cells defective for p53 function.

Apoptosis↗
PubMed1995

Dominant oncogenes, tumor suppressors, and radiosensitivity.

A variety of conflicting results appeared in the literature concerning the effect of dominant oncogenes on the sensitivity to irradiation and to anticancer agents in a number of cell lines of human and animal origin. In this report we provide evidence supporting the hypothesis that the tumor suppressor gene p53 and the apoptosis suppressor gene bcl2 modulate the effect of dominant oncogenes and that the effect of dominant oncogenes on resistance or sensitivity is dependent on the balance between the expression of p53 and bcl2.

Apoptosis↗
PubMed1994

Bcl-2 overexpression abolishes early calcium waving preceding apoptosis in NIH-3T3 murine fibroblasts.

Overexpression of the apoptosis protection gene bcl-2 abolished the earliest response to serum withdrawal in NIH-3T3 murine fibroblasts, i.e., the abrupt cytoplasmic free calcium drop. This phenomenon, also observed in a myeloid cell line, led us to propose this ionic waving as an early apoptotic signal. Its abolition by bcl-2 overexpression suggests that this gene plays a role also on early events "priming" apoptosis.

3T3 Cells↗
PubMed1994

Apoptosis and the cell cycle.

This brief review examines the strict relationships between cell apoptosis and G1 cyclins. It has been shown that the basic role of G1 cyclins is in regulating G1 progression and G1/S transition (the critical cycle point for cell program decisions, including apoptosis) a fatal program for cells unable to bypass G1/S checkpoint 1. Notably, both of the two giant regulators of checkpoint 1 (i.e., p105RB [retinoblastoma oncosuppressor-encoded protein] and p53 dependent WAF1/CIP1) are influenced by or influence G1 cyclins: cyclin E/cdk2 kinase complexes hyperphosphorylate p105RB, induce E2F release, and free G1 exit. On the other hand, p21-WAF1/CIP1 is an inhibitor of cyclin-dependent kinases blocking cells at G1/S. Thus, G1 cyclin activity appears as a conditio sine qua non for G1 exit and apoptosis escape.

Animals↗
PubMed1993

Apoptosis induction in 32D cells by IL-3 withdrawal is preceded by a drop in the intracellular calcium level.

The 32D murine myeloid cell line is dependent on interleukin-3 (IL-3) for growth in vitro. We show here that IL-3 induces a slow increase in endocellular calcium, which is sizeable two hours after the addition. Withdrawal of the cytokine induces apoptosis, whose classic late events are evident after 16/18 hours, and are preceded by a calcium drop during the first 2/3 hours after IL-3 subtraction. Calcium drop is here proposed as a trigger of the apoptotic process, in agreement with other recently reported findings.

Animals↗
PubMed1993

Negative growth control by a novel low M(r) phosphotyrosine protein phosphatase in normal and transformed cells.

Having determined the complete amino acid sequence of a cytosolic phosphatase purified from bovine liver, we studied the role of this enzyme (referred to as 'PTPase') in the control of cell proliferation. We used NIH/3T3 fibroblasts, both normal and transformed by the oncogenes v-erbB, v-src, and v-raf: a synthetic gene coding for PTPase was transfected into, and overexpressed in, normal and transformed NIH/3T3 cells with resulting inhibition of cell growth. Inhibition of proliferation correlated with the level of foreign PTPase; growth in soft agar was also inhibited in transformants overexpressing the enzyme. However, PTPase overexpression did not inhibit the rapid turnover of inositol lipids stimulated by platelet-derived growth factor. We conclude that this novel PTPase is active on cell type-specific signalling substrates that control normal and transformed fibroblast proliferation.

3T3 Cells↗
PubMed1993

Cytokine receptor signal transduction mechanisms in immuno-hematopoietic cells.

Novel aspects of cytokine receptor signal transduction are discussed and cytokine receptors classified based on ligand-dependent signalling. An introductory section presents an overview of the role of cytokines in hematopoiesis. A brief explanation of basic concepts, such as redundancy, pleiotropy, synergism, etc., important for the understanding of cell response to cytokines, is also included. Three of five classes of receptors show the involvement of tyrosine kinase activity as the key step in signal transduction. The importance of tyrosine phosphorylation in cellular response to cytokines is pointed out.

Animals↗
PubMed1992

Overexpression of a synthetic phosphotyrosine protein phosphatase gene inhibits normal and transformed cell growth.

We studied the level of the cytosolic phosphotyrosine protein phosphatase (PTPase) (originally termed low-M(r) acid phosphatase) in normal NIH/3T3 and in v-erbB-transformed fibroblasts. The level of the enzyme, assayed by ELISA, was inversely related to cell proliferation, normally growing cells had less enzyme than their contact-inhibited counterparts and v-erbB transformants had less enzyme than normal NIH/3T3. In order to overexpress the enzyme and study its effects in normal and transformed cells, we transfected a synthetic gene coding for the PTPase in control NIH/3T3 and v-erbB transformants. The overexpressed enzyme was recognized by antibodies raised against the native enzyme and, in cells overexpressing the PTPase, we observed a marked dephosphorylation of tyrosyl residues of cellular proteins. Cell proliferation, in both normal and v-erbB transformants overexpressing the PTPase, was measured. We observed that PTPase overexpression was accompanied by significantly reduced thymidine incorporation in both cell types, either serum-starved or serum-stimulated. The ability of transformed v-erbB cells to grow in soft agar was also markedly decreased by overexpression of the enzyme. Taken together, our results indicate that overexpression of PTPase might interfere with mitogenic signalling pathways in both normal and transformed cells, and propose a role for PTPase in the control of cell proliferation.

3T3 Cells↗
PubMed1992

Mitogenic signal transduction: a common target for oncogenes that induce resistance to ionizing radiations.

We hypothesized that resistance to ionizing radiations accompanying neoplastic transformation caused by some oncogenes was due to common biochemical pathways affecting the mechanism of mitogenic signal transduction. In order to verify this hypothesis, we studied the formation of mitogenic second messengers in cells transformed by oncogenes that induce radioresistance. We observed an increase of diacylglycerol which activates protein kinase C, an increase of phosphatidylcholine metabolism, with a concomitant decrease of inositol lipid metabolism. Our data show that sensitivity to ionizing radiations was inversely related to the intracellular level of diacylglycerol; study of signalling alterations in spontaneous tumors could provide predictive indications about the responsiveness of neoplasia to radiation therapy.

3T3 Cells↗
PubMed1992

Changes in pyridine and adenine nucleotide levels in Friend erythroleukaemia cells during growth and differentiation.

Pyridine and adenine nucleotide levels were measured in Friend erythroleukaemia cells (FELC) stimulated to growth and induced to differentiate by hexamethylene bisacetamide (HMBA) and N'-methylnicotinamide (N'-MNAM). A three- to fourfold increase in the NADP(H) was found to parallel cell growth stimulation in both the presence and absence of differentiation inducers. NAD(H) increased about twofold in control and to a minor extent in HMBA-treated FELC but did not vary significantly in N'-MNAM-treated cells. ATP was significantly higher in control cells stimulated to growth than in resting ones, but it did not vary in inducer-treated cells. These data confirm the relationship between high NADP(H) levels and cell resumption to growth; moreover they show that NAD(H) pool reduction and NAD/NADH ratio rise are associated with the process of FELC differentiation. The activities of NAD pyrophosphorylase and NAD kinase are much more enhanced in growth-stimulated FELC than in resting ones. On the other hand transition from the quiescent to the proliferative state was accompanied by a decrease in the activity of poly(ADP-ribose) polymerase. A decrease in poly(ADP-ribose) polymerase activity was also found in differentiated cells in contrast to controls.

Acetamides↗
PubMed1991

Gold labeling of urokinase plasminogen activator. Characterization and specific binding in cultured mammalian cells.

A colloidal gold-urokinase plasminogen activator complex (u-PAGC) was prepared and characterized. It was used as an ultrastructural marker to study binding sites for urokinase in human and dermal fibroblasts and bovine adrenal endothelial cells in culture. Both the preparation conditions for 15 nm in diameter gold particles and their labeling with urokinase molecules are reported. The complex was stable for at least 4 weeks and had efficient binding and biological activity. Colloidal gold conjugate was observed as single particles or small clusters scattered on the plasma membrane of the cells at 0 degree C and within vesicles in the cytoplasm after a few minutes at 37 degrees C. These data suggest that urokinase-gold complex is a useful marker for the specific labeling of urokinase binding sites.

Adrenal Glands↗
PubMed1990

Transformation by ras oncogene induces nuclear shift of protein kinase C.

We measured protein kinase C (PKC) activity in normal and ras-transformed Balb/3T3 fibroblasts; cytosolic and nuclear-associated PKC activity was determined either as phorbol ester binding, PKC-dependent phosphorylation of histone III-S, or phosphorylation of endogenous nuclear proteins. Results demonstrate that ras-transformed fibroblasts show down-regulation of cytosolic PKC accompanied by increase of nuclear-associated PKC. These results provide evidence linking transformation to PKC nuclear shift with consequent phosphorylation of nuclear proteins.

Animals↗
PubMed1990

Interaction between endogenous circulating sulfated-glycosaminoglycans and plasma proteins.

Interaction between endogenous 35S-labelled plasma glycosaminoglycans and proteins in murine plasma was demonstrated by coelution from gel chromatography of circulating 35S-labelled glycosaminoglycans with a wide range of plasma proteins. Autoradiography of electrophoretic tracing of proteins from 35S-sulfate labelled plasma showed that labelled glycosaminoglycans were associated with alpha 1, alpha 2, beta globulins and albumin, but not with gamma globulins. Analysis by gel chromatography on Sepharose CL-6B of delipidated 35S-labelled plasma after either proteolysis or beta-elimination, suggested that 35S-labelled glycosaminoglycan chains were covalently bound to proteins. Lipids were probably involved in the supramolecular assembly of GAGs with plasma proteins, as shown by hydrophobic interaction chromatography. In addition, strong, non-covalent interaction between glycosaminoglycan chains and proteins was responsible for the difficulty in extracting 'free' glycosaminoglycans from plasma. Consistently, ion-exchange chromatography of 35S-sulfate labelled delipidated plasma after alkali treatment, revealed that the anionic properties of glycosaminoglycans were hampered when plasma proteins were present.

Animals↗
PubMed1990

Interaction of urokinase A chain with the receptor of human keratinocytes stimulates release of urokinase-like plasminogen activator.

On the basis of a fibrinolytic assay with 125I-fibrin, zymography, and immunoprobing with anti-human urokinase antibody, we have observed that the in vitro established NCTC human keratinocyte cell line releases into the culture medium a 54,000-Da plasminogen activator which is indistinguishable from human urokinase. Only the early release following the washing of keratinocyte monolayers is accounted for by secretion of preformed enzyme, while late secretory events require the de novo synthesis of urokinase. The released enzyme can interact by autocriny with its own receptor present on keratinocytes. The addition to the keratinocyte culture medium of the urokinase A chain can stimulate a concentration-dependent urokinase oversecretion, which is not paralleled by oversecretion of plasminogen activator inhibitor-1. Since stimulation of urokinase production can be obtained by an A chain concentration (5 ng/ml) which was previously shown to be efficient in inducing keratinocyte mobilization in an in vitro migration model system, we hypothesize that this mechanism may be important in vivo during the process of wound repair.

Cell Line↗
PubMed1990

Role of specific membrane receptors in urokinase-dependent migration of human keratinocytes.

On the basis of both 125I-labeled plasminogen activator binding analysis and transmission electron microscopy studies of the interaction of a plasminogen activator/gold complex with cell membranes, we have found that human keratinocytes have specific receptors for human urokinase-type plasminogen activator distributed on the cell surface as singlets, or as small or large clusters. The use in binding experiments of the purified A chain of urokinase-plasminogen activator and of anti-A chain monoclonal antibodies has indicated that cell receptors are specific for a sequence present on the A chain, as previously reported for other cells. The interaction of both the native molecule and the purified A chain with such receptors stimulates mobilization of keratinocytes in an in vitro cell model system (Boyden chamber), when present in the lower compartment of the migration apparatus in nanomolar concentrations. Preincubation of chemoattractants with a monoclonal antibody which prevents receptor/ligand interaction also prevents plasminogen activator-induced cell migration. These data suggest that, under the conditions used in this in vitro model system, the plasminogen activator-dependent mobilization of keratinocytes depends on the interaction of the ligand with free receptors on the cell surface, and is independent of plasmin generation.

Antibodies, Monoclonal↗
PubMed1990

Modulation of urokinase receptors on human synovial cells and osteoarthritic chondrocytes by diacetylrhein.

On the basis of both 125I-labelled urokinase-like plasminogen activator binding analysis and transmission electron microscopy of an urokinase-gold complex, we have shown the presence of specific receptors for human urokinase on the cell membrane of human synovial cells. By radio-ligand binding experiments we have shown the existence of similar receptors on the surface of human chondrocytes. In both cases the specific binding is attributable to interaction between the receptor and the A chain of the ligand, as previously shown in other cell model systems. Treatment of synoviocytes with 1,8-diacetoxy-anthraquinone-3-carboxylic acid (diacetylrhein) is able to reduce the number of surface urokinase receptors. At the same time the drug can reduce the fibrinolytic activity released into the culture medium of human synovial cells. Preliminary data indicate that chondrocytes from osteoarthritic patients have a larger number of urokinase receptors than chondrocytes of normal patients. Diacetylrhein can restore the receptor number to normal levels; the amount of urokinase in the synovial fluid of ostroarthritic patients is also reduced. We conclude that the use of this drug has the chance to significantly modify the natural history of osteoarthritis.

Anthraquinones↗
PubMed1989

Synthesis of diacylglycerol de novo is responsible for permanent activation and down-regulation of protein kinase C in transformed cells.

We measured the synthesis of diacylglycerol de novo in normal NIH/3T3 fibroblasts and in cells transformed by ras, src, sis and abl oncogenes. Analysis of the incorporation of glucose-derived 14C into diacylglycerol indicated that neosynthesis of diacylglycerol was constitutively active in the transformed cell lines. Elevated levels of diacylglycerol and persistent activation/down-regulation of protein kinase C reduced the binding of phorbol dibutyrate to transformed cells. This phenomenon could be reversed by blocking the glycolytic pathway, thus indicating that neosynthesized diacylglycerol was responsible for persistent activation and down-regulation of protein kinase C. In transformed cells, protein kinase C activity could not be stimulated by the addition of diolein; however, inhibition of glycolysis restored the ability of transformed cells to respond to diolein. Taken together these data indicate that constitutive synthesis of diacylglycerol de novo is responsible for activation and down-regulation of protein kinase C in transformed cells, and it may play a role in altered mitogenic signalling.

Animals↗