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Biomedical subjects

M Fanciulli

Publications and source records attributed to M Fanciulli.

At least 37 records · Page 2Linked to original sources
PubMed1994

Defective human retinoblastoma protein identified by lack of interaction with the E1A oncoprotein.

Inactivating mutations of the retinoblastoma susceptibility gene (Rb) are involved in the pathogenesis of hereditary and sporadic retinoblastoma. Alterations in the Rb gene have also been found in several other human tumors occurring with epidemiological incidence higher than that of retinoblastoma. Four human malignant glioma cell lines were examined for abnormalities in the retinoblastoma gene product (pRb), using a procedure based on the interaction of pRb with an in vitro-translated adenovirus E1A oncoprotein. In the CRS-A2 cell line, derived from a glioblastoma multiforme, pRb did not bind with the in vitro-translated E1A protein. Restriction analysis of the CRS-A2 Rb gene and Rb mRNA expression provided patterns that could not be distinguished from the other glioma cell lines. Further investigation revealed the presence of a truncated pRb in the CRS-A2 cell line, due to a nucleotide insertion in the coding sequence at position 2550. In addition, this truncated Rb protein was undetectable in phosphorylated form. The binding assay with the in vitro-translated E1A was also used to study other cell lines with known mutations in the Rb gene. This method, which evaluates the interaction between in vitro-translated E1A and the pRb, is proposed as a rapid screening for detecting functional alterations in the retinoblastoma protein.

Adenovirus E1A Proteins↗
PubMed1994

Glycolysis and growth rate in normal and in hexokinase-transfected NIH-3T3 cells.

The glycolytic enzyme hexokinase plays a key role in regulating cell energy metabolism. Its activity has been associated with cell growth rate and, notably, with neoplastic transformation. NIH-3T3 cells were transfected with a tumor hexokinase cDNA. The transfected cells showed increased hexokinase amount and activity, mainly located in the particulate cellular fraction, increased glycolytic rate evaluated as lactate production, and, finally, enhanced growth rate. These data may suggest that high hexokinase activity might be not merely the consequence of peculiar metabolic demands by actively replicating normal or cancer cells, but also a modification able per se to drive, at least partially, a more intense mitotic activity.

3T3 Cells↗
PubMed1994

Effect of the local anesthetic bupivacaine on the energy metabolism of Ehrlich ascites tumor cells.

The effect of the local anesthetic bupivacaine on the energy metabolism of Ehrlich ascites tumor cells has been investigated. Even at low concentrations, bupivacaine decreased the oxygen uptake rate, but its effect was remarkably higher on the uncoupled respiration. Experiments on specific segments of the respiratory chain have shown that bupivacaine did not inhibit electron transport from Q to oxygen. Spectroscopic evidences demonstrated a NAD(P)H oxidation in bupivacaine-treated cells respiring on endogenous substrates, indicating that the inhibition of oxygen depended on a reduced electron transport from site 1-entering substrates to respiratory chain. The aerobic glycolysis was stimulated by low and inhibited by high bupivacaine concentrations. The increased lactate production rate was due to an activation of mitochondrial ATPase, whereas its decrease was related to an inhibition of the hexokinase activity.

Adenosine Triphosphate↗
PubMed1993

Rhein inhibits glucose uptake in Ehrlich ascites tumor cells by alteration of membrane-associated functions.

Rhein (RH), 4,5 dihydroxyanthraquinone-2-carboxylic acid, is known to inhibit the glycolysis of neoplastic cells by impairing glucose uptake. In order to establish whether this might be due to a selective interaction of the carrier with the drug or to functional modifications of the cell membrane, the effect of RH on glucose uptake in Ehrlich ascites tumor cells has been investigated. RH strongly inhibits the uptake of both 2-deoxyglucose and 3-O-methylglucose, so the reduced influx therefore cannot be ascribed to an effect on glucose phosphorylation. The inhibition of glucose transport does not depend on a reduction of the number of the carriers as indicated by the inability of the drug to interfere with the synthesis of the transporter. Moreover, the extent of total binding of cytochalasin B, as well as the fact that glucose specificity is not altered, indicate that the intrinsic activity of the glucose carrier is not affected. We therefore conclude that the inhibition of glucose uptake must be ascribed to an interaction of the drug with cell membranes that results in an alteration of membrane-associated functions.

Amino Acid Sequence↗
PubMed1993

Glucose metabolism in adriamycin-sensitive and -resistant LoVo human colon carcinoma cells.

The utilization of carbon from 14C-labeled glucose by adriamycin (ADM)-sensitive and -resistant LoVo human colon carcinoma cells has been investigated. The following summarizes the results: 1. Aerobic glycolysis is the main energy-yielding process in both cell lines, whereas only a small proportion of glucose carbon atoms are incorporated into CO2, lipids, nucleic acids, and supporting structures. 2. Basic alterations in glucose metabolism are associated with drug resistance in tumor cells. In fact, ADM-resistant LoVo cells show a significant increase in the oxidative pathway of glucose metabolism as well as in acetyl-CoA production. 3. In adriamycin-resistant LoVo cells, the amount of glucose carbon atoms metabolized through the pentose phosphate pathway is significantly higher than in adriamycin-sensitive cells. These findings confirm a modified glucose metabolism in cells with a resistant phenotype.

Adenocarcinoma↗
PubMed1992

Inhibition of membrane redox activity by rhein and adriamycin in human glioma cells.

The effect of the combination of adriamycin (ADM) with the anti-inflammatory drug rhein (RH) on the membrane redox activity in human glioma cells was investigated. RH, although less effective than ADM, inhibits ferricyanide reduction by human glioma cells in a dose-dependent manner as well as ferricyanide-induced proton release. The inhibition of the plasma membrane redox system might represent a further mechanism by which RH, other than ATP depletion, affects cell survival. The analysis of the interaction between ADM and RH, performed with the isobolar method, demonstrates a strong synergic response, probably due to an effect on different sites of action. The synergism of the ADM-RH association allows us to achieve a pre-established extent of inhibition with ADM concentrations much lower than with ADM alone. RH might, therefore, represent a very useful tool to improve the therapeutic index of ADM and to lower its general toxicity.

Anthraquinones↗
PubMed1991

Glucose utilization by tumor cells: a post-translational modification of mitochondrial hexokinase may play a regulatory role.

In Northern blot analysis of a series of tumor cell lines a single hexokinase mRNA species of 4.3 Kb was detected. Detailed examination of one such line, the rat AS-30D hepatoma, revealed that two mitochondrial species of hexokinase are present with a molecular mass of 115 and 107 KDa. The smaller of the two species is 4-fold more active than the larger. Only the larger, less active species is detected in the well differentiated H-35 rat hepatoma cell line which exhibits a lower glucose catabolic rate. These results suggest that a post-translational proteolytic event may play a central role in regulating the glucose utilization capacity of tumor cells by modulating the relative levels of high and low activity forms of hexokinase.

Animals↗
PubMed1990

Glucose phosphorylation in tumor cells. Cloning, sequencing, and overexpression in active form of a full-length cDNA encoding a mitochondrial bindable form of hexokinase.

In rapidly growing tumor cells exhibiting high glucose catabolic rates, the enzyme hexokinase is markedly elevated and bound in large amounts (50-80% of the total cell activity) to the outer mitochondrial membrane (Arora, K.K., and Pedersen, P.L. (1988) J. Biol. Chem. 263, 17422-17428; Parry, D.M., and Pedersen, P.L. (1983) J. Biol. Chem. 258, 10904-10912). In extending these studies, we have isolated a cDNA clone of hexokinase from a lambda gt11 library of the highly glycolytic, c37 mouse hepatoma cell line. This clone, comprising 4,198 base pairs, contains a single open reading frame of 2,754 nucleotides which encode a 918-amino acid hexokinase with a mass of 102,272 daltons. This enzyme exhibits, respectively, 68 and 32 amino acid differences, including several charge differences, from the recently sequenced human kidney and rat brain enzymes. The putative glucose and ATP binding domains present in the latter two enzymes and in rat liver glucokinase are conserved in the tumor enzyme. At its N-terminal region, tumor hexokinase has a 12-amino acid hydrophobic stretch which is present in the rat brain enzyme but absent in the rat liver glucokinase, a cytoplasmic enzyme. The mature tumor hexokinase protein has been overexpressed in active form in Escherichia coli and purified 9-fold. The overexpressed enzyme binds to rat liver mitochondria in the presence of MgCl2. This is the first report describing the cloning and sequencing of a tumor hexokinase, and the first report documenting the overexpression of any hexokinase type in E. coli. Questions pertinent to the enzyme's mechanism, regulation, binding to mitochondria, and its marked elevation in tumor cells can now be addressed.

Amino Acid Sequence↗
PubMed1990

Glucose phosphorylation. Interaction of a 50-amino acid peptide of yeast hexokinase with trinitrophenyl ATP.

A 50-amino acid peptide predicted by chemical modification studies of yeast hexokinase to contain an ATP-binding site has been synthesized and purified. The peptide, which includes residues from glutamate 78 at the NH2-terminal end to leucine 127 at the COOH-terminal, resides within the smaller of the two lobes found in the three-dimensional structure of yeast hexokinase. It is this region which has been reported recently to exhibit significant sequence homology with hexokinase types I and IV of higher eukaryotic cells and sequence homology with the active site of protein kinases. Similar to native yeast hexokinase, the 50-amino acid peptide interacts strongly with the fluorescent analog TNP-ATP [2',(3')-O-(2,4,6-trinitrophenyl)-adenosine-5'-triphosphate]. A 5-fold enhancement is observed when 8 microM peptide interacts with 20 microM TNP-ATP. The stoichiometry of binding is very close to 1 mol of TNP-ATP/mol peptide. Also, similar to native yeast hexokinase, the fluorescent enhancement observed upon TNP-ATP binding to the synthetic peptide is greater than that observed upon TNP-ADP binding. Finally, TNP-AMP exhibits a much lower fluorescent enhancement in the presence of hexokinase or the synthetic peptide. The additional findings that ATP can readily prevent TNP-ATP binding and that TNP-ATP can substitute for ATP as a weak substrate for hexokinase in the phosphorylation of glucose indicate that the synthetic peptide described here comprises part of the catalytic site.

Adenosine Triphosphate↗
PubMed1989

rp-120: a common endogenous substrate for insulin and IGF-1 receptor-associated tyrosine kinase activity in the highly malignant AS-30D rat hepatoma cells.

Receptors for Insulin, Epidermal Growth Factor, Platelet-Derived Growth Factor and Insulin-like Growth Factor type 1 are tyrosine-specific protein kinases. This enzymatic activity may play a role in mediating the biological actions of these peptides. It has recently been identified a Mr 120 KDa glycoprotein in rat liver plasma membranes which can be phosphorylated by the insulin receptor and by the EGF receptor in a cell-free system and by the insulin receptor in intact cultured H-35 hepatoma cells. In the present report it is shown that the solubilized Insulin-like Growth Factor type 1 receptor can phosphorylate tyrosine residues in the same 120 KDa glycoprotein from the AS-30D rat hepatoma cells.

Animals↗
PubMed1988

Effect of lonidamine on human malignant gliomas: biochemical studies.

Lonidamine (LND) has been shown to inhibit tumor aerobic glycolysis. Its effect was evaluated on several human astrocytomas at different degrees of malignancy; a correlation was found between LDN effect on lactate production and tumor malignancy: in grade I and II astrocytomas LND stimulates lactate production, while in grade III, IV and glioblastoma multiforme lactate production is inhibited. In an attempt to explain this different behaviour, hexokinase content and compartmentation was evaluated in astrocytomas from fresh operatory specimens and from cultured cells as well, observing a significative correlation between malignancy, hexokinase activity, percent of mitochondrially-bound hexokinase and LND effect. The results justify from a biochemical point of view the role of LND as a 'non-conventional' agent in multimodality combined treatment for malignant gliomas.

Astrocytoma↗
PubMed1987

Effect of hyperthermia on electron transport in Ehrlich ascites tumor mitochondria.

The effect of hyperthermia (1 hr, 41 degrees C) on the functional properties of Ehrlich ascites tumor mitochondria was investigated. Mitochondria isolated from Ehrlich ascites tumor after exposure of whole cells to 41 degrees C for 1 hr still phosphorylate and maintain a normal acceptor control ratio (ACR). The temperature decreases state 4 and ADP-and FCCP-stimulated respiration on various substrates entering at three energy-conserving sites of the respiratory chain. The inhibition of oxygen consumption by NAD- and FAD-linked substrates was 40% for state 4 and 70% for ADP- or FCCP-stimulated respiration. State 4 and FCCP-stimulated respiration of mitochondria on TMPD + ascorbate was affected 38% and 45%, respectively. ATPase activity was unaffected by hyperthermia, indicating that under these experimental conditions, the inhibition of ADP-stimulated respiration does not depend on an effect on either Fo F1-ATPase or adenine translocase, the activity of which is required for ATP entry prior to ATPase activity. Because of the inability to detect a specific site of action of temperature, it is conceivable that hyperthermia might inhibit substrate oxidation by altering some components of the inner mitochondrial membrane, which regulates the kinetic properties of the membrane-associated enzymes.

Adenosine Triphosphatases↗