Biomedical subjects

E Prosperi

Publications and source records attributed to E Prosperi.

63 records · Page 4Linked to original sources

PubMedDate not supplied

Influence of daunorubicin on membrane permeability properties: detection by means of intracellular accumulation and efflux of fluorescein.

The influence of daunorubicin (DNR) on membrane permeability properties was assessed by studying the ability of living HeLa cells to exhibit fluorochromasia; that is, to take up a fluorogenic substrate and retain the fluorescent compound obtained by enzymatic reaction. The intracellular accumulation of the fluorescent product as well as its release by the cells may be considered indicators of the permeability properties, since both processes are mediated by the cell membrane. The influence of the drug on the accumulation and on the efflux of fluorescein, obtained intracellularly from the hydrolysis of fluorescein diacetate (FDA), was evaluated, after DNR treatment, by measuring the fluorescence intensity of the product in single living cells by flow cytometry. The results showed that DNR, up to a concentration of 5 X 10(-6) M, did not significantly affect the accumulation of fluorescein. On the contrary, the efflux was strongly inhibited. A comparative study of the influence of drugs with known action mechanism was performed with the membrane-active compound hydrocortisone (HC) and with the metabolic inhibitor KCN. The results obtained indicate that DNR significantly affects membrane permeability properties and that its influence is similar to that exerted by metabolic inhibitors.

Cell Membrane Permeability↗

PubMedDate not supplied

Poly(ADP-ribose) synthesis: a useful parameter for identifying apoptotic cells.

Cell death by apoptosis was analysed in HeLa cells either treated with the antitumoral drug bleomycin or depleted of growth factors by long-term culture without medium change. The interference of apoptosis with normal cell cycle progression was followed by flow cytometry in cells stained with propidium iodide and with antibody to S-phase-related PCNA protein. Bleomycin-treated cells showed a net accumulation in G2/M phase paralleled by the appearance of material with a subdiploid DNA content. Cells with a subdiploid DNA content were also present in growth factor-depleted cultures and were shown to derive from all the cell cycle phases. To identify apoptotic features in HeLa cell cultures, we applied a recently developed assay based on the simultaneous analysis in the single cell of three parameters, namely chromatin condensation, DNA degradation and poly(ADP-ribose) synthesis. Apoptotic cells were visualized by sequential reactions: Hoechst staining, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labelling assay and immunoreaction with anti-poly(ADP-ribose) monoclonal antibody. Positive reactions were obtained for cells at different stages of the apoptotic programme showing condensed nuclei, fragmented chromatin and apoptotic bodies.

Apoptosis↗

PubMedDate not supplied

Effects of topoisomerase II inhibitors on gastric cancer cells characterized by different genetic lesions.

Gastric cancer is poorly-responsive to widely used antitumour drugs, the efficacy of which is thought to be related to the capacity of triggering apoptosis. This process requires a series of gene products including a functional p53 protein. We tested the effects of two DNA topoisomerase II poisons, etoposide and doxorubicin, on gastric cancer cell lines with different genetic lesions. We characterised MKN74 and MKN28 cells for p53 gene status and for the expression of p53 and p21 proteins, as well as of topoisomerase II alpha and beta isoforms. After drug treatments, the cells were analysed for drug cytotoxicity, colony forming ability, cell cycle distribution and presence of apoptotic features. Our findings demonstrated that both etoposide and doxorubicin have a potent anti-proliferative effect on gastric cancer cells. Cell death kinetics was different in the two cell lines, MKN74 cells being more sensitive than MKN28 to the drugs. MKN74 cells, although harboring a wt p53 gene, were unable to undergo a massive apoptosis following etoposide treatment. The response of this cell line might be related to the topoisomerase II beta isozyme, the expression of which proved to be undetectable.

Antibiotics, Antineoplastic↗

PubMedDate not supplied

Topoisomerase II alpha and beta in human tumor cells grown in vitro and in vivo.

The cellular content and the cell-cycle distribution of the 170 kD- and 180 kD-isoforms of DNA topoisomerase II were investigated in human tumor cells with specific monoclonal antibodies and by immunofluorescent detection with flow cytometry. Levels of topo II alpha were almost three-fold higher than the beta-isozyme in exponentially growing cells in vitro. In contrast, topo II alpha but not beta, was markedly reduced in plateau-phase cells. Tumor cells from surgical biopsies, mainly in G0/G1 phase, exhibited a 95% beta- versus 5% alpha-isoform expression. These results support the hypothesis that topo II alpha is mainly related to DNA synthesis, and topo II beta to DNA transcription.

Antibodies, Monoclonal↗

PubMedDate not supplied

Metabolic energy availability and proliferative activity in adriamycin-treated cells.

Metabolic energy availability and proliferative activity were studied in HeLa cells treated with Adriamycin (Adr). Conditions (ID50) that induced growth delay, without significant cell lethality, did not appreciably alter ATP content, over 72 hours after treatment. Energy-dependent membrane permeability properties, assessed with the vital dye fluorescein, were similarly unaffected. The cytostatic effect was confirmed by the incorporation rate of 3H-thymidine and 14C-leucine, and by flow cytometric analysis of cell cycle progression. Early depletion in cellular ATP occurred when Adr induced an evident cytocidal effect.

Adenosine Triphosphate↗

PubMedDate not supplied

Doxorubicin resistance circumvention by verapamil in B16 melanoma cells.

We present data on the cellular drug pharmacokinetic alterations correlated with the circumvention of doxorubicin resistance by verapamil in a B16 melanoma cell line. An increased drug uptake, a decreased drug efflux and a different intracellular drug distribution appear to be responsible for the enhancement of doxorubicin cytotoxicity induced by treatment with verapamil in drug-resistant cells. However, doxorubicin pharmacokinetics and cytotoxicity were not affected by verapamil in doxorubicin-sensitive melanoma cells.

Animals↗

PubMedDate not supplied

DNA-protein cell content of lymphokine activated killer (LAK) and target cells in coculture.

An extensive study of the interactions between LAK-target cells in cocultures was performed by means of cytometric (relative DNA and protein content) and morphological parameters. The aim was to obtain new information about the cell cycle stage of tumor target cells (Chang line) during the attack of LAK cells. Specimens obtained from cocultures at 0, 24, 48 hours were processed for electron microscopy, flow cytometry, immunocytochemistry (anti-BrdU reaction) and light microscopy (L.I., M.I., cell-cell interaction). The most intense activity of LAK cells against the target was found at 24 hours of coculture independently of the proliferative stage of the tumor cells. These results suggest that the high affinity between killer and target cells was not affected by the molecular changes of the Chang cell surface during the cell cycle.

Cell Communication↗

PubMedDate not supplied

Free radical-dependent DNA lesions are involved in the delayed cardiotoxicity induced by adriamycin in the rat.

The role of free radicals in the genesis of adriamycin (ADR)-induced delayed cardiotoxicity and the cardioprotective effects of the spin trap N-tert-butyl-alpha-phenylnitrone (PBN) were investigated in an in vivo rat model. As ADR and free radicals are no longer present in the myocardium by the time the delayed effects of the drug become apparent, ADR has been proposed to act by causing early radical-dependent DNA lesions, resulting in impaired synthesis of critical target proteins. DNA lesions were detected 10 days after ADR treatment (3X3 mg/kg i.v.) and were still present at the time of onset of the delayed cardiomyopathy. PBN, administered by a slow-release osmotic pump to maintain constant plasma levels throughout the time of persistence of ADR in the myocardium (approximately 2 weeks), prevented the development of DNA lesions, as well as the late contractile and electrical impairment induced by the anthracycline, thus supporting the hypothesis that free radicals play a causal role in both phenomena.

Animals↗

PubMedDate not supplied

The spin trap alpha-phenyl-tert-butyl nitrone protects against myelotoxicity and cardiotoxicity of adriamycin while preserving the cytotoxic activity.

The possibility of preventing the myelo- and cardiotoxicity of adriamycin (ADR), was explored in in vivo experiments in the rat. Assuming that free radicals play a key role in the ADR organotoxicity, a new anti-radical approach was set up by administering a spin trapping compound (PBN) which is taken up by the cells and specifically interacts with radicals. ADR was given i.v., 3 mg/kg every 3rd day for 3 times. PBN was continuously administered throughout the persistence time of ADR in myocardium (15 days). Serial ECG and leukocyte counting were performed during 10 weeks, then hearts were isolated and Langendorff-perfused; functional parameters (heart rate, contractility, coronary flow) were evaluated. PBN improved ECG and prevented the myelotoxicity, while functional parameters were not significantly different from those of control. Cytotoxicity was evaluated in vitro in 3 different human tumour cell lines; PBN did not modify the cytotoxicity of ADR, thus excluding a free radical involvement in this activity. The present results suggest that a proper administration schedule of spin traps might be a promising approach for improving the therapeutic index of ADR.

Animals↗