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Biomedical subjects

M Donzelli

Publications and source records attributed to M Donzelli.

16 recordsLinked to original sources
PubMed2001

Different effects of methotrexate on DNA mismatch repair proficient and deficient cells.

Antifolates exert their antiproliferative activity through the inhibition of dihydrofolate reductase and, as a consequence, of thymidylate synthesis, thereby inducing nucleotide misincorporation and impairment of DNA synthesis. We investigated the processes involved in the repair of antifolate-induced damage and their relationship with cell death. Since misincorporated bases may be removed by DNA mismatch repair (MMR), the study was carried out on the MMR-proficient human cell lines HeLa and HCT116+chr3, and, in parallel, on the MMR-deficient cell lines HeLa cell-clone12, defective in the protein hPMS2, and HCT116, with an inactive hMLH1. After treatment with methotrexate (MTX), we observed that DNA repair synthesis occurs independently of the cellular MMR function. Clear signs of apoptosis such as nuclear shrinkage, chromatin condensation and degradation, DNA laddering, and poly (ADP-ribose) polymerase (PARP) proteolysis, were visible in both MMR(+) and MMR(-) cells. Remarkably, cell viability was lower and the apoptotic process was triggered more efficiently in the MMR-competent cells.

Antimetabolites, Antineoplastic↗
PubMed1999

Apoptosis-prone phenotype of human colon carcinoma cells with a high level amplification of the c-myc gene.

Although apoptosis can be induced by the enforced expression of exogenously introduced c-myc genes, it is not clear whether overexpression resulting from the amplification of the resident c-myc gene in tumor cells is sufficient to induce apoptosis. We have investigated the relationship between c-myc gene amplification and the propensity of tumor cells to undergo apoptosis, using the SW613-12A1 and SW613-B3 cell lines, which are representatives, respectively, of tumorigenic and non-tumorigenic clones isolated from the SW613-S human colon carcinoma cell line. Tumorigenic clones are characterized by a high level of amplification and expression of the c-myc gene, whereas cells of non-tumorigenic clones have a small number of copies and a lower level of expression of this gene. Analysis of c-myc mRNA level in cells cultured under low serum conditions indicated that the expression of the gene is tightly regulated by serum growth factors in non-tumorigenic B3 cells, whereas it is poorly regulated in tumorigenic 12A1 cells, the level of mRNAs remaining relatively high in serum-starved 12A1 cells. Under these conditions, 12A1 cells showed clear evidence of apoptosis, whereas B3 cells were completely refractory to the induction of apoptosis. Moreover, the study of cell lines derived from non-tumorigenic apoptosis-resistant clones following the introduction by transfection of exogenous c-myc gene copies showed that they have acquired an apoptosisprone phenotype. Altogether, our results strongly suggest that deregulated c-myc expression due to high-level amplification confers an apoptosis-prone phenotype to tumor cells. The possible consequences of these observations for cancer therapy are discussed.

Clone Cells↗
PubMed1997

Multiparametric staining to identify apoptotic human cells.

To analyze relevant features of HeLa and HL60 cells driven into apoptosis by etoposide, we have developed a new "tricolor" assay, based on the simultaneous analysis in the single cell of chromatin condensation, DNA degradation, and cellular poly(ADP-ribose) synthesis. The latter reaction is catalyzed by poly(ADP-ribose)-polymerase (E.C. 2.4.2.30), an enzyme which is activated by the presence of DNA free ends. The protocol consists in the visualization of apoptotic cells by Hoechst staining, TUNEL assay, and immunoreaction with anti-poly(ADP-ribose) antibody. We thus provide the first evidence that endogenous poly(ADP-ribose) production is indeed stimulated in cells undergoing apoptosis after treatment with antitumoral drugs, and that the monitoring of this endogenous enzymatic reaction, combined with morphological and other biochemical parameters, should facilitate the detection of apoptotic cells.

Adenosine Diphosphate Ribose↗
PubMed1996

Sequence of events leading to apoptosis in long term cultured HeLa cells.

We have maintained HeLa cells in culture in the original medium for increasing times to induce growth arrest. Cell viability was evaluated by trypan blue dye exclusion assay. We observed that when cells are maintained in culture for several days, morphological hallmarks of apoptosis become evident. DNA synthesis rate, followed by (3)H-thymidine incorporation slowed down in long term cultured cells. This evidence was supported by the analysis of cell cycle progression determined by proliferating cell nuclear antigen (PCNA) immunostaining. Apoptotic cells have been characterized with respect to the sequential appearance of high molecular weight and internucleosomal DNA fragmentation. We have provided evidence that in this experimental model the first step in DNA degradation is represented by the formation of high molecular weight fragments, whereas nucleosomal DNA ladder is visible later on. The activation of the enzyme poly(ADP-ribose)polymerase, considered a marker of apoptotic death, has been observed. The results suggest that long term culture conditions activate the apoptotic programme.

Journal Article↗
PubMed1995

Induction of apoptotic cell death by DNA topoisomerase II inhibitors.

We have analyzed the interference of antitumoral drugs acting through the inhibition of DNA topoisomerase II on the human HeLa cell metabolism. Different compounds characterized by a diverse mechanism of action have been used, namely m-amsacrine, an intercalative drug, etoposide, which does not intercalate DNA, and suramin, which exerts its effect through an unknown mechanism. In HeLa cells treated with increasing doses of these drugs, we have examined cell viability and DNA synthesis capacity, and we have evaluated topoisomerase II activity. Cellular morphology and DNA integrity have been studied in order to characterize the mechanism of cell death. The results we have obtained clearly indicate that topoisomerase II poisons induce cell death by apoptosis. These observations suggest a role of the inhibition of topoisomerase II activity in the apoptotic program.

Amsacrine↗
PubMed1995

Activation of poly(ADP-ribose)polymerase in apoptotic human cells.

We have studied the effect of the chemotherapeutic drug VP-16 (etoposide) on the metabolism of HeLa cells by analysing different cellular parameters considered as markers of apoptosis. Typical features such as chromatin condensation and internucleosomal DNA cleavage are visible in HeLa cells exposed to VP-16. We investigated whether the appearance of small-sized DNA fragments could regulate the ADP-ribosylation process. To this purpose, we have analysed, by means of the activity gel technique; the structural and catalytical properties of poly(ADP-ribose)polymerase. In extracts from cells where etoposide-induced DNA fragmentation occurred, we have shown that the label of the autoribosylated form of the enzyme is greatly increased even if the amount of the protein remains constant. This phenomenon is completely abolished in cells preincubated with poly(ADP-ribose)polymerase inhibitor, 3-aminobenzamide. After VP-16 administration, we have observed that the level of NAD is not heavily decreased. It is widely agreed that zinc exerts an inhibitory effect on the endonuclease(s) responsible for the fragmentation of DNA during apoptosis. After incubation of cells with zinc/VP-16 we have found the occurrence of apoptotic parameters even in the absence of internucleosomal DNA cleavage. The inhibition of DNA fragmentation prevents the activation of poly(ADP-ribose)polymerase activity. These results indicate that the activation of the enzyme towards the automodification reaction is strictly dependent on the appearance of DNA internucleosomal fragments and could represent a way to control enzyme activity.

Adenosine Monophosphate↗
PubMed1994

Dose-dependent zinc inhibition of DNA ladder in apoptotic HeLa cells regulates the activity of poly(ADP-ribose) polymerase and does not protect from death induced by VP-16.

Zinc ions exert an inhibitory effect on Ca(2+)Mg(2+)-dependent endonuclease which is supposed to be responsible for the fragmentation of DNA during apoptosis. In the experimental system we used, that is HeLa cells treated with VP-16, the protection from internucleosomal DNA degradation is modulated by Zn concentration and appears to be dependent on the time after treatment. This effect does not prevent cell death or occurrence of apoptotic parameters, suggesting that DNA ladder appearance is not a crucial event in apoptosis. The activation of poly(ADP-ribose)polymerase following the administration of VP-16, is not observed in cells in which DNA fragmentation has been abolished by zinc, supporting the hypothesis that this event is regulated by the appearance of small-sized DNA fragments.

Journal Article↗
PubMed1990

[Mitral valve replacement in young children: a long-term follow-up].

The Authors report follow-up data (mean 60, range 13-101 months) from 4 patients under two years of age (mean 11.5 months) operated on for prosthetic mitral valve implantation. Two patients were male and two female. Pre-operative diagnosis was partial atrioventricular septal defect with parachute mitral valve in one case and isolated mitral valve anomaly in the others (1 parachute, 2 hammock valves). One bioprosthesis (Liotta n. 23) and three mechanical prostheses (Björk-Shiley) were implanted. Complications occurred in two patients: one case of endocarditis on the bioprosthesis, leading to substitution with a Björk-Shiley valve; one case of acute thrombosis 8 months after implantation in the only pt receiving aspirin as prophylactic therapy. This complication was resolved by intravenous thrombolytic agents. All the patients are alive, in I NYHA functional class and receiving oral anti-coagulants. Instrumental follow-up with eco-Doppler demonstrated normal functioning prostheses in all the patients. A sub-aortic gradient was found in the patient with partial atrioventricular septal defect. Prosthesis-patient mismatch was identified in a patient operated on when she was 6 months old who was followed-up for 5 years.

Bioprosthesis↗
PubMed1983

Supraventricular tachycardia in infants: use of the "diving reflex".

The effectiveness of the "diving reflex" in treating paroxysmal supraventricular tachycardia (PSVT) in 10 infants aged under 6 months is demonstrated. No rhythm disturbances were encountered during the maneuver, even in patients receiving digitalis. The recommended water temperature is 5 degrees C and the total time of facial immersion, between 6 and 7 seconds. The diving reflex might be considered the first therapeutic approach in infants under 6 months old with PSVT.

Electrocardiography↗
PubMed1981

[Persistence of fetal circulation. Clinical and angiographic evaluation of two cases. Review of the literature (author's transl)].

Persistent fetal circulation ("PFC") is a condition characterized by perduring after birth of fetal hemodynamic pattern: persistence of high pulmonary vascular resistance then occurs. "PFC" is a high mortality syndrome recently recognized as a cause of severe cyanosis and tachypnea in full-term newborns. Perinatal hypoxia, hyperviscosity, acidosis etc, may be also responsible of a persistence of high pulmonary vascular resistance. The increased right heart pressures cause a right to left shunt across the ductus arteriosus and foramen ovale not yet completely closed at birth. In this paper the clinical and angiographic evaluation of two full-term newborns, males, 5 days and 5 months old, are described, who presented with tachypnea, cyanosis and heart failure. Moreover we want to make a critical review of medical literature about this subject. The electrocardiogram of both patients showed right ventricular hypertrophy and myocardial ischemia. The chest roentgenograms showed cardiac enlargement and increased pulmonary vascular markings. Lung and central nervous system disorders were excluded by clinical and instrumental exams. Both patients underwent cardiac catheterization and angiocardiography in order to exclude a congenital heart disease. There was just evidence of pulmonary vascular resistance and right to left shunt at ductal and atrial level. O2 100% administration in one patient determined a decrease in pulmonary resistance and the shunting abolition. Clinical and hemodynamic patterns of this syndrome are very different. There are "simple forms" with normal myocardial function and "complex" higher mortality forms with evidence of cardiac involvement. It is often difficult to distinguish such forms from the other causes of central cyanosis and/or cardiac failure in the newborn as transposition of the great arteries, total anomalous pulmonary venous connection: the individualization of the syndrome then may be impossible without cardiac catheterization. A correct diagnosis for a precocious and reasonable treatment are extremely important for the survival of such critical newborns.

Angiocardiography↗
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

[Kawasaki's disease. Presentation of 2 clinical cases].

The authors describe two cases of Kawasaki disease, one of whose is an "incomplete" form, and emphasize the importance to know this "new" disease in the differential diagnosis of febrile exanthemas of childhood. A complete diagnostic walk-up failed to find out a probably, while change of some immunological and flogistic parameters has permitted to suppose a probably immuno-mediated pathogenesis of Kawasaki disease. Both patients healed without any sequelae after therapy with low doses of salicylate for ten months, in confirmation of usefulness of this treatment in the prevention of heart complications of Kawasaki disease.

Aspirin↗