Biomedical subjects

G Citro

Publications and source records attributed to G Citro.

At least 37 records · Page 2Linked to original sources

PubMed1996

Effect of cisplatin and c-myb antisense phosphorothioate oligodeoxynucleotides combination on a human colon carcinoma cell line in vitro and in vivo.

We investigated the effect of c-myb antisense phosphorothioate oligodeoxynucleotides [(S)ODNs] and cisplatin (CDDP) combination on the human colon carcinoma cell line LoVo Dx both in vitro and in nude mice bearing LoVo Dx solid tumour. We show that antisense (S)ODN treatment decreases c-myb mRNA and protein expression, induces growth arrest in the G1 phase of the cell cycle, and inhibits cell proliferation. In vivo treatment with c-myb antisense (S)ODNs results in a reduction in tumour growth. A greater inhibition of cell proliferation in vitro and a higher increase of tumour growth inhibition and growth delay in vivo were obtained with the combination of (S)ODNs and CDDP than when the two agents were administered separately. This comparative study, using the same tumour cell line in vitro and in vivo, suggests that c-myb antisense (S)ODNs might be useful in the therapy of colon cancer in combination with antineoplastic drugs.

Animals↗

PubMed1996

Effect of the antitumor drug lonidamine on glucose metabolism of adriamycin-sensitive and -resistant human breast cancer cells.

The effect of lonidamine on glucose metabolism, hexokinase activity and adenylate pool of MCF-7 human breast cancer cells sensitive and resistant to adriamycin has been investigated. The following summarizes the results: 1. In both cell types the greatest part of glucose was metabolized to lactate, whereas only a small proportion of glucose carbon atoms was incorporated into CO2, lipids, nucleic acids, and supporting structures. 2. Glucose utilization, lactate production, and ATP content were higher in resistant cells due to a greater activity of mitochondrial hexokinase. 3. Lonidamine decreased glucose utilization, aerobic glycolysis and ATP content in both cell types and the effect was significantly higher on resistant cells. 4. The extent of inhibition in sensitive and resistant cells overlapped that found for mitochondrially bound hexokinase, thus indicating that the greater sensitivity of resistant cells to lonidamine was due to their higher amount of bound hexokinase. These findings confirmed a modified glucose metabolism in cells with resistant phenotype and suggested that lonidamine might be usefully used to reduce or overcome multidrug resistance of those cells with a reduced ability to accumulate and retain antitumor drugs.

Adenine Nucleotides↗

PubMed1996

Liposomal targeting of leukaemia HL60 cells induced by transferrin-receptor endocytosis.

A liposomal carrier system able to interact specifically with HL60 leukaemia cells was produced using small unilamellar liposomes made of pure phospholipids chemically cross-linked to human transferrin. The conjugation of transferrin to liposomes was carried out using N-succinimidyl 3-(2-pyridyldithio)-propionate and 2-iminothiolane as activating agents for the liposomes and the protein. The reaction occurred under conditions set to covalently link on the surface of a single vesicle a limited number (one to ten) of transferrin molecules, as verified by means of electron microscopy and immunoenzymic measurements. Before conjugation, the ultrastructure of the liposomes, and the content and distribution of the amino groups within the bilayer, were determined. The reactivity of the liposomes towards amino-derivatizing or thiolating compounds was also measured. Kinetic spectroscopic measurements confirmed that the distribution of the phosphatidylethanolamine in the vesicle bilayer is asymmetrical: 22% of phosphatidylethanolamine was found exposed to the external surface of the liposomes and accessible to the cross-linker. The modified liposomes were able to interact specifically with the cells and to be internalized by active receptor-mediated endocytosis, as demonstrated by the full inhibition of internalization induced by free transferrin.

Endocytosis↗

PubMed1995

A modified spectroscopic method for the determination of the transbilayer distribution of phosphatidylethanolamine in soya-bean asolectin small unilamellar vesicles.

A spectroscopic kinetic approach for determining the relative concentrations of phosphatidylethanolamine (PE) exposed on the external and internal layers of small unilamellar vesicles (SUVs) used as a model system and prepared by sonication of purified soya-bean asolectin is proposed, based on the use of 2,4,6-trinitrobenzenesulphonic acid (TNBS) and N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP). The known reactions between PE and TNBS and/or SDPD were used, separately or in combination, to derivatize PE in preformed vesicles. We have observed that mixing SUVs with excess TNBS results in a biphasic time course. Kinetic analysis of the data supports the conclusion that external PE is rapidly derivatized (fast phase) with a half-time of 2 min. In the next (slow) phase (half-time 70 min), TNBS permeates the vesicle membrane and also reacts with PE molecules facing the internal liposomal compartment. Under the experimental conditions chosen, SPDP reacted with only the external PE molecules. The reaction of SUVs first derivatized with SPDP and then with TNBS further demonstrates that the two phases, observed with TNBS, are due to modification of external and internal PE. Approx. 30% of PE was found to be facing the external bulk phase, thus confirming the asymmetric distribution of the molecules in SUVs. The maximum number of thiol arms covalently linked by means of SPDP modification of PE on the surface of a single liposome was estimated at about 10(2).

Cross-Linking Reagents↗

PubMed1995

Monomeric Pseudomonas aeruginosa nitrite reductase: preparation, characterization, and kinetic properties.

Monomeric nitrite reductase in an active form has been prepared by controlled succinylation of the dimeric native enzyme of Pseudomonas aeruginosa and subsequent purification. The monomeric enzyme has an optical spectrum indistinguishable from that of the native enzyme. On the other hand, circular dichroic spectra in the heme and peptide absorption regions show differences with respect to the dimer that indicate that the chemical modification and/or the dissociation into monomers somewhat perturb the chromophores' environment and the secondary structure. The (negatively charged) monomer is unable to oxidize its physiological substrates, azurin and cytochrome c551. This loss of activity is not due to monomerization, but is linked to the total net charge of the succinylated molecule, which interestingly enough acquires the ability to oxidize efficiently eukaryotic cytochrome c (which is not a substrate of the native dimeric enzyme). Stopped-flow studies show that the reduced monomer reacts with oxygen with a kinetic pattern similar to that shown by the dimeric enzyme. However, a higher reaction rate in the bimolecular binding of oxygen and a much higher oxygen affinity than for the native enzyme are observed. The evidence reported in this paper indicates that the dimeric state of Pseudomonas nitrite reductase is not a prerequisite for the ferrocytochrome c-oxygen oxidoreductase activity of this enzyme.

Circular Dichroism↗

PubMed1995

Identification of gains and losses of DNA sequences in primary bladder cancer by comparative genomic hybridization.

Comparative genomic hybridization (CGH) makes it possible to detect losses and gains of DNA sequences along all chromosomes in a tumor specimen based on the hybridization of differentially labeled tumor and normal DNA to normal human metaphase chromosomes. In this study, CGH analysis was applied to the identification of genomic imbalances in 26 bladder cancers in order to gain information on the genetic events underlying the development and progression of this malignancy. Losses affecting 11p, 11q, 8p, 9, 17p, 3p, and 12q were all seen in more than 20% of the tumors. The minimal common region of loss in each chromosome was identified based on the analysis of overlapping deletions in different tumors. Gains of DNA sequences were most often found at chromosomal regions distinct from the locations of currently known oncogenes. The bands involved in more than 10% of the tumors were 8q21, 13q21-q34, 1q31, 3q24-q26, and 1p22. In conclusion, these CGH data highlight several previously unreported genetic alterations in bladder cancer. Further detailed studies of these regions with specific molecular genetic techniques may lead to the identification of tumor suppressor genes and oncogenes that play an important role in bladder tumorigenesis.

Chromosome Mapping↗

PubMed1994

Lonidamine-mediated respiratory changes in rat heart myocytes: a re-examination of the functional response of mitochondrial cytochrome c oxidase.

Respiratory activity of intact cardiac myocytes isolated from rats treated with lonidamine (LND) has been examined under conditions where cytochrome oxidase turns over at its maximal rate. Compared to myocytes isolated from control rat hearts, those treated with LND displayed a 60% increase in the cytochrome oxidase-dependent rate of respiration; electron microscopy revealed, in agreement with the literature, that the membrane structure of the mitochondrion had become disorganized. The increase in the rate of oxygen consumption was correlated with the (partial) impairment of the membrane ability to maintain the proton electrochemical potential gradient which normally inhibits oxidase activity. Results are discussed with reference to previous reports showing no effect of LND on cytochrome c oxidase activity. The evidence reported better clarifies the contribution of cytochrome oxidase to the demonstrated energetic failure displayed by cells treated with LND.

Animals↗

PubMed1994

Wild-type p53 gene expression induces granulocytic differentiation of HL-60 cells.

Overexpression of wild-type p53 gene in malignant cell lines has been shown to inhibit cell proliferation in a number of cases. However, endogenous p53 protein seems to play little role in normal cell-cycle control as suggested by the normal development of p53 null mice, and by the low p53 protein levels expressed in most cell types. Recently, increased expression of endogenous p53 protein has been observed during the cellular response to DNA damage, as well as during differentiation of human hematopoietic cells. To study the role of the p53 gene in hematopoietic differentiation, we introduced the wild-type p53 gene or the temperature-sensitive p53(Val135) mutant into p53-deficient HL-60 promyelocytic leukemia cells. Morphological analysis, flow-cytometric determination of granulocytic or monocytic surface markers, and ability to reduce nitroblue tetrazolium (NBT) demonstrated that expression of exogenous wild-type p53 gene in HL-60 cells induces differentiation through the granulocytic pathway. Proliferation and cell-cycle analysis performed early after expression of wild-type p53 showed that induction of differentiation is not coupled with growth arrest, which suggests that p53 is involved in differentiation independently of its activity on the cell cycle.

Base Sequence↗

PubMed1994

Effects of sequential combinations of N-methylformamide with adriamycin on cultured melanoma cells (M14).

The effects of sequential combinations of N-methylformamide (NMF) with adriamycin (ADM) on a human melanoma cell line (M14) were investigated. The results obtained can be summarized as follows: (i) When NMF was administered before ADM (NMF-->ADM), a decrease of ADM-induced cytotoxicity and intracellular ADM content was revealed. Conversely, the reverse combination (ADM-->NMF) produced a remarkable reduction of cell survival accompanied by a significant intracellular drug retention. (ii) The fluorescence microscopy revealed that in NMF- and in NMF-->ADM-treated cells the actin microfilament distribution appeared to be very similar to that in control cells. On the contrary, a disorganization of the microfilament architecture was observed in ADM-->NMF-treated cells. (iii) The microtubule organization was significantly altered by NMF. This effect appeared to be even more evident in M14 cells treated with the combination ADM-->NMF. (iv) Confocal microscopy observations showed an intracytoplasmic retention of ADM in ADM-->NMF-treated cells. These findings suggest that the cytoskeletal changes induced by NMF might interfere with the ADM efflux mechanisms, accounting for the high drug level and low survival detected in ADM-->NMF-treated cells.

Antineoplastic Agents↗

PubMed1994

Chemical modification of ligands for cell receptors to introduce foreign compounds into the cells.

PURPOSE: In all forms of drug therapy, clinicians must ensure that the maximum therapeutic benefit is achieved without inducing unacceptable toxicity. An improvement in the therapeutic index could be achieved through the targeting of drugs selectively to cancer cells. METHODS: We have developed a delivery system that uses receptor-mediated endocytosis to introduce oligodeoxynucleotides into cells bearing receptors for the ligands used as a vehicle. Human transferrin, as well as folic acid and steroid, has been covalently linked to polylysines of various sizes through a disulfite bridge and used as oligomer carriers. RESULTS AND DISCUSSION: The inhibitory effect of c-myb antisense oligodeoxynucleotides conjugate to modified transferrin on LoVo Dx cell proliferation was examined. Protocols to modify physiologic ligands to be used as vehicles for a selective delivery are shown. Modified ligand molecules should also be used to covalently bind liposome-carrying compounds able to affect neoplastic growth.

Cells, Cultured↗

PubMed1994

Inhibition of leukaemia cell proliferation by folic acid-polylysine-mediated introduction of c-myb antisense oligodeoxynucleotides into HL-60 cells.

The inhibitory effect of c-myb antisense oligodeoxynucleotides (ODNs) conjugated to folic acid (FA) on HL-60 cell proliferation was examined. Folic acid was covalently linked to a polylysine chain and purified by gel chromatography. Sterile FA-polylysine was complexed with c-myb sense and antisense. Exposure of HL-60 cells to the FA-polylysine-c-myb antisense ODN complex resulted in a down-regulation of c-myb expression and a greater inhibition of proliferation than that obtained using free ODNs. Moreover, FA-polylysine conjugate alone or complexed to c-myb sense ODN was not toxic to cells. The antigenic properties and uptake of the vitamin were not affected by the polylysine chain. These data suggest that this strategy is potentially useful for the selective delivery of anti-oncogene-targeted ODNs into cancer cells.

Base Sequence↗

PubMed1994

Intracellular localization of the antitumour drug adriamycin in living cultured cells: a confocal microscopy study.

The intracellular distribution of the anthracyclinic antibiotic adriamycin in living cultured cells has been investigated by confocal microscopy. In human melanoma cells (M14), adriamycin was localized inside the nuclei. When adriamycin-treated M14 cells were allowed to recover in drug-free medium, a complete efflux of the drug from the nucleus was revealed. In recovered cells, a weakly fluorescent signal was observed in the perinuclear region. When M14 cells were recovered in a medium containing colcemid, a microtubule depolymerizing agent, the drug transport from the nucleus to the cell periphery appeared to be inhibited, suggesting that the microtubule network is strongly involved in drug transport mechanisms. In multidrug-resistant (MDR) cells the intracellular location of adriamycin was shown to be noticeably different from that of the parental wild-type cells. In particular, in resistant human breast carcinoma cells (MCF-7), adriamycin appeared to be exclusively located within the cytoplasm whereas the nuclei were shown to be completely negative. When adriamycin treatment was performed in association with MDR revertants, such as Lonidamine (inhibitor of the energy metabolism) or verapamil (inhibitor of the P-glycoprotein efflux pump), a marked enhancement of the cytoplasmic signal was observed in resistant cells. Under these conditions, adriamycin appeared concentrated in the perinuclear region, but the nuclei were still negative. Confocal microscopy proved to be a very useful method for the study of the intracellular transport of fluorescent substances, such as anthracyclinic antibiotics, and for the investigation of the multidrug resistance phenomenon in tumour cells.

Breast Neoplasms↗

PubMed1994

Antiapoptotic effect of c-fes protooncogene during granulocytic differentiation.

The c-fes protooncogene is expressed at high levels in the terminal stages of granulocytic differentiation. Its product, p92c-fes, exhibits a tyrosine-kinase activity and is involved in the cellular response to GM-CSF, but its role is not yet clarified. To study this problem, the c-fes protooncogene expression has been inhibited in HL60 cells and in fresh leukemic blast cells of Acute Promyelocytic Leukemia (APL) induced to differentiate with All-Trans-Retinoic Acid (ATRA). Inhibition of c-fes function was obtained by treatment of the cells with a specific antisense oligomer complementary to the 5' region of the c-fes mRNA. It was observed that the cells, rather then differentiate to granulocytes, underwent premature cell death showing the morphological and molecular characteristics of apoptosis. Superimposable results are obtained on blast cells from APL. It is possible to conclude that the loss of cell viability that occurs during the in vitro differentiation of myeloid cells, after the complete inhibition of c-fes expression and treatment with ATRA, is due to activation of programmed cell death rather than an accelerated differentiation. Our data suggest that the c-fes product is essential for the survival of myeloid cells during differentiation.

Apoptosis↗

PubMed1993

Inhibition of c-fes expression by an antisense oligomer causes apoptosis of HL60 cells induced to granulocytic differentiation.

The c-fes protooncogene is expressed at high levels in the terminal stages of granulocytic differentiation, but so far no definite function has been attributed to the product of this oncogene. To tackle this problem, the c-fes protooncogene expression has been inhibited in HL60 cells, and fresh leukemic promyelocytes of acute promyelocytic leukemia have been induced to differentiate with retinoic acid (RA) and dimethylsulfoxide (DMSO). Inhibition was obtained by incubating the cells with a specific c-fes antisense oligodeoxynucleotide. It was observed that the cells, rather than differentiating, underwent premature cell death showing the morphological and molecular characteristics of apoptosis. This process was inhibited by granulocyte and granulocyte/macrophage colony-stimulating factor, but not by interleukin 3 (IL-3), IL-6, or stem cell factor. Our present results demonstrate that the loss of cell viability that occurs during the in vitro differentiation of myeloid cells, after the complete inhibition of the c-fes gene product and treatment with RA-DMSO, is due to activation of programmed cell death. It is concluded that a possible role of the c-fes gene product is to exert an antiapoptotic effect during granulocytic differentiation.

Apoptosis↗

PubMed1993

Activation of 2,4-diaminotoluene to proximate carcinogens in vitro, and assay of DNA adducts.

1. 2,4-Diaminotoluene, which yields adducts with DNA in vivo, has been studied for its ability to form adducts in vitro. Metabolic activation with rat liver post-mitochondrial supernatant gave 300 adducts/10(6) nucleotides in calf thymus single-stranded DNA, under defined experimental conditions. 2. 2,4-Diaminotoluene-modified DNA and deoxyhomopolymers showed characteristic u.v. absorption spectra, exhibiting hyperchromic effects at 235 and 220 nm, and hypochromic effect at 260 nm. The difference spectra between diamine-modified and untreated DNA, or deoxyhomopolymer, were very similar to the spectrum of 2,4-diaminotoluene alone. 3. 2,4-Diaminotoluene-modified DNA was assayed by ELISA with specific monoclonal antibodies directed against diamine-DNA adducts. Reactions with poly-d(A) or poly-d(A-T) gave no spectral modification, and immunochemical analysis showed that the diamine did not bind to these polynucleotides. On the other hand, in the case of poly-d(G) or poly-d(C-G), strong immunoreactions were observed, demonstrating that the guanine base is involved in the binding of the diamine to DNA. 4. Monoclonal antibodies directed against different diamine-DNA adducts have shown that 80% of the in vitro metabolic activation involves the para amino group of the aromatic diamine.

Animals↗

PubMed1993

Receptor-mediated oligodeoxynucleotides delivery by estradiol and folic acid polylysine conjugates.

The lack of efficient and specific delivery to target cells still limits the potential application of antisense oligodeoxynucleotides as therapeutic agents in cancer disease. We have covalently linked a polylysine chain (10,000-20,000 mw) to compounds as folic acid, retinoic acid, transferrin, insulin and estradiol, to deliver c-myb antisense oligonucleotide into tumor cells. Using these complexes as carriers for the oligodeoxynucleotides can be achieved an increase in their uptake into target cells through a natural endocytosis pathway.

Biological Transport, Active↗

PubMed1993

Release of cytochromes from hypoxic and reoxygenated guinea pig heart.

Isolated, perfused hearts from guinea pigs were subjected to hypoxia for 30 minutes followed by reoxygenation for 30 minutes. Cellular damage was assessed by measuring the release of the cytoplasmic enzyme lactate dehydrogenase and the mitochondrial markers cytochrome c and cytochrome oxidase. The release of the enzymes was correlated with electron microscopy. Hypoxia induced an increase in the release of lactate dehydrogenase and cytochrome c. During reoxygenation, the release of lactate dehydrogenase was exacerbated while that of cytochrome c decreased, suggesting a partial recovery of the mitochondria. Cytochrome oxidase was not detectable in the extracellular space during hypoxia or reoxygenation. It is suggested that cytochrome c is a specific marker for damage to mitochondria caused by hypoxia and its loss may affect respiratory chain function.

Animals↗

PubMed1992

Inhibition of leukemia cell proliferation by receptor-mediated uptake of c-myb antisense oligodeoxynucleotides.

Exposure of human leukemia HL-60 cells to an oligodeoxynucleotide complementary to an 18-base sequence (codons 2-7) of c-myb-encoded mRNA has previously been shown to result in inhibition of cell proliferation. Because HL-60 cells express high levels of transferrin receptor we adapted a DNA delivery system based on receptor-mediated endocytosis to introduce myb oligomers complexed with a transferrin-polylysine conjugate into those cells. A DNA.RNA duplex resistant to S1 nuclease digestion was detected as early as 12 hr after culture of HL-60 cells in the presence of the myb antisense/transferrin-polylysine complex. Exposure of HL-60 cells to the myb antisense/transferrin-polylysine complex resulted in rapid and profound inhibition of proliferation and loss of cell viability much more pronounced than that occurring in cells exposed to free myb antisense oligodeoxynucleotides. The transferrin-polylysine/myb sense complex or the transferrin-polylysine conjugate alone had no effect on HL-60 cell proliferation and viability. These findings indicate that myb synthetic oligodeoxynucleotides enter efficiently into HL-60 by transferrin receptor-mediated endocytosis and exert a profound biological effect. Such a delivery system could exploit other ligand-receptor interactions for the selective delivery of oncogene-targeted antisense oligodeoxynucleotides.

Antineoplastic Agents↗