[Obesity and Eating Disorders. Indications for the different levels of care. An Italian Expert Consensus Document]. / Documento di Consensus. Obesità e Disturbi dell'Alimentazione Indicazioni per i diversi livelli di trattamento.

This paper is an Italian Expert Consensus Document on multidimensional treatment of obesity and eating disorders. The Document is based on a wide survey of expert opinion. It presents, in particular, considerations regarding how clinicians go about choosing the most appropriate site of treatment for a given patient suffering from obesity and/or eating disorders: outpatient, partial hospitalization, residential rehabilitation centre, inpatient hospitalization. In a majority of instances obesity and eating disorders are long-term diseases and require a multiprofessional team-approach. In determining an initial level of care or a change to a different level of care, it is essential to consider together the overall physical condition, medical complications, disabilities, psychiatric comorbidity, psychology, behaviour, family, social resources, environment, and available services. We first created a review manuscript, a skeleton algorithm and two rating scales, based on the published guidelines and the existing research literature. As the second point we highlighted a number of clinical questions that had to be addressed in the specific context of our National Health Service and available specialized care units. Then we submitted eleven progressive revisions of the Document to the experts up to the final synthesis that was approved by the group. Of course, from point to point, some of the individual experts would differ with the consensus view. The document can be viewed as an expert consultation and the clinical judgement must always be tailored to the particular needs of each clinical situation. We will continue to revise the Document periodically based on new research information and on reassessment of expert opinion to keep it up-to-date. The Document was not financially sponsored.

Etoposide induces the dispersal of DNA ligase I from replication factories.

In eukaryotic cells DNA replication occurs in specific nuclear compartments, called replication factories, that undergo complex rearrangements during S-phase. The molecular mechanisms underlying the dynamics of replication factories are still poorly defined. Here we show that etoposide, an anticancer drug that induces double-strand breaks, triggers the redistribution of DNA ligase I and proliferating cell nuclear antigen from replicative patterns and the ensuing dephosphorylation of DNA ligase I. Moreover, etoposide triggers the formation of RPA foci, distinct from replication factories. The effect of etoposide on DNA ligase I localization is prevented by aphidicolin, an inhibitor of DNA replication, and by staurosporine, a protein kinase inhibitor and checkpoints' abrogator. We suggest that dispersal of DNA ligase I is triggered by an intra-S-phase checkpoint activated when replicative forks meet topoisomerase II-DNA--cleavable complexes. However, etoposide treatment of ataxia telangiectasia cells demonstrated that ataxia-telangiectasia-mutated activity is not required for the disassembly of replication factories and the formation of replication protein A foci.

p21waf1/cip1 protein associates with the detergent-insoluble form of PCNA concomitantly with disassembly of PCNA at nucleotide excision repair sites.

Evidence is presented that after exposure of normal human fibroblasts to UV-C light, nuclear binding of the proliferating cell nuclear antigen (PCNA) required for nucleotide excision repair, was rapidly triggered in the G1 and G2 phases of the cell cycle. Association to repair sites of the detergent-insoluble form of PCNA reached a peak 15-30 min after irradiation, and then decreased to basal levels within 24-48 h. In contrast, the nuclear association of p21 protein showed a slower kinetics, reaching maximal levels between 24 and 48 h but, similarly to PCNA, occurring only in G1 and G2 phases. Although the two proteins are known to be associated as detergent-soluble proteins, it is unknown whether they associate also in the detergent-insoluble form. To address this question, the chromatin-bound form of PCNA was released by using DNAse I. DNA digestion resulted in the almost complete release of PCNA from its binding sites, while only about 60% of nuclear-bound p21 could be solubilized. Immunoprecipitation of PCNA and p21 released by enzymatic digestion showed that p21 was associated with PCNA bound to late DNA repair sites. These results indicate that during nucleotide excision repair, nuclear binding of PCNA precedes that of p21 protein, and suggest that temporal association of p21 with the detergent-insoluble fraction is coincident with the disassembly of PCNA from DNA repair sites.

p21(waf1/cip1)-null human fibroblasts are deficient in nucleotide excision repair downstream the recruitment of PCNA to DNA repair sites.

The cyclin-dependent kinase inhibitor p21(waf1/cip1) is known to impair DNA synthesis by binding to PCNA, the co-factor of DNA polymerases delta and epsilon. However, a positive role for p21 in nucleotide excision repair (NER) has been suggested. In this study, the sensitivity to DNA damage and DNA repair efficiency were investigated in p21-null human fibroblasts obtained by targeted homologous recombination. After UV-C irradiation, p21-/- cells showed a threefold reduction in clonogenic survival and an increased susceptibility to apoptosis, as compared with parental p21+/+ cells. Removal of cyclobutane pyrimidine dimers was significantly reduced in p21-/- cells both in the whole genome, and at the level of the rDNA gene cluster, as determined by immunoassay and Southern blot, respectively. After DNA damage, the recruitment of PCNA as detergent-insoluble form associated to DNA repair sites in p21-/- fibroblasts, was comparable to that observed in parental p21+/+ cells. However, PCNA remained associated with DNA for a longer period in p21-/- than in p21+/+ cells. These results suggest that in human cells, p21 is required for NER at a step located downstream the recruitment of PCNA to DNA repair sites.

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.

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.

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.

Differential involvement of DNases in HeLa cell apoptosis induced by etoposide and long term-culture.

We have applied to human HeLa cells two different stimuli of apoptosis: the antitumoral drug etoposide, and a more 'physiological' death condition, obtained by growing cells in the same medium for long time periods, for up to 10 days. Analysis of different parameters demonstrated that in both experimental systems the same apoptotic features are visible. However, the DNA degradation pattern appeared to be different, suggesting the involvement of different DNases. In this view, we have analyzed the activity and expression of Ca2+-Mg2+-dependent and acid DNases. We have observed that DNase I is not modulated during apoptosis. In contrast, the acid L-DNase II (derived from Leukocyte Elastase Inhibitor by post-translational modification), recently identified in our laboratory, is mainly active in the apoptotic pathway induced by long term-culture. Furthermore, we have provided evidence that while caspase 3 is activated by both inducers, caspase 1 is essential only for the etoposide-induced apoptosis.

Expression of the 170-kDa and 180-kDa isoforms of DNA topoisomerase II in resting and proliferating human lymphocytes.

The expression of the 170-kDa (alpha) and the 180-kDa (beta) isoforms of DNA topoisomerase II (topo II) was investigated with two specific monoclonal antibodies (MoAbs) in human peripheral blood lymphocytes (PBL), before and after phytohaemoagglutinin (PHA) stimulation. Binding of each MoAb was detected by indirect immunofluorescence labelling and quantified with flow cytometry. In resting PBL, the intensity of immunostaining was very low for both isozymes; however, topo IIbeta-associated immunofluorescence was about 2.5 times significantly higher (P<0.001) than that associated with the alpha isoform. Between 48 and 72 h of PHA stimulation, when the highest percentage of cells in S and G2+M phases was found, the levels of topo IIalpha and beta increased up to about 30 and 10 times the value measured in resting PBL, respectively. Thus, the two isoforms reached comparable immunofluorescence values. At longer stimulation periods (96-120 h), topo IIalpha immunofluorescence was not significantly changed, while that relative to topo IIbeta declined to about 50% of the peak value (P<0.02). At this time however, topo IIalpha-associated immunofluorescence was not significantly different from that related to the beta isozyme. These results suggest that in resting PBL topo IIalpha is required at levels lower than topo IIbeta, while in proliferating lymphocytes both isoforms are expressed to significantly higher levels.

Comparative studies on the effects of doxorubicin and differentiation inducing agents on B16 melanoma cells.

The differentiation-inducing activity of doxorubicin on B16 melanoma cells grown in vitro was compared with that of other known differentiation inducers, such as theophylline, retinoic acid, and melanocyte-stimulating hormone (MSH). At drug concentrations resulting in cytostatic effects, doxorubicin and theophylline induced morphological changes (dendritic-like structures with a terminal melanin granule) with an enhancement of total melanin content and tyrosinase activity. Retinoic acid did not alter melanin content and cell morphology, although it affected cell growth. MSH enhanced total melanin content and tyrosinase activity, with no significant morphological changes. Flow cytometric analysis showed that MSH led to an accumulation of cells in G1 phase whereas doxorubicin induced an accumulation of cells in G2 + M. Studies on DNA content in doxorubicin-treated cells, selected on the basis of a morphologically differentiated pattern, showed a clustering of these cells in G2 + M, probably due to a cytokinesis block. Thus doxorubicin can induce cell differentiation comparable with other differentiation inducers.

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.

Nuclear binding of cell cycle-related proteins: cyclin A versus proliferating cell nuclear antigen (PCNA).

We have investigated the cell cycle-dependent nuclear binding of cyclin A and of the proliferating cell nuclear antigen (PCNA) in asynchronously growing human fibroblasts. To this purpose, we have applied flow cytometry immunofluorescence, a powerful technique for elucidating the cell cycle phase during which the nuclear binding occurs. We have observed that, in striking contrast with the distribution of nuclear-bound PCNA which is restricted to S phase, the immunofluorescence signal of the nuclear-bound form of cyclin A is high in the G1 and G2 phases of the cell cycle. These results suggest the involvement of nuclear-bound cyclin A in the G1/S and G2/M phase transitions.

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.

Early effects of AZT on mitochondrial functions in the absence of mitochondrial DNA depletion in rat myotubes.

Zidovudine (AZT) is a potent inhibitor of human immunodeficiency virus (HIV) replication. In humans, as well as in animal models, long-term treatment with AZT induces a severe myopathy characterised by structural and functional alterations of mitochondria associated with depletion of mitochondrial DNA (mtDNA). In the present work, we compared the effects induced by AZT on mitochondria upon short- or long-term treatments of cultured rat myotubes. Morphological alterations were investigated by electron microscopy, and mtDNA depletion and deletions were analysed by Southern blot. Mitochondrial membrane potential was determined after JC-1 staining by laser-scanning confocal microscopy in whole cells, and by flow cytometry in isolated muscle mitochondria. We found that the early effects of AZT on mitochondrial functions were a marked, yet reversible reduction in mitochondrial membrane potential, in the absence of any effect on mtDNA. The long-term treatment, in addition to mitochondrial membrane potential alterations, induced morphological changes in mitochondria, and a remarkable reduction in the amount of mtDNA, without any significant evidence of mtDNA deletions. In both treatments, a block of the spontaneous contraction of myotubes was observed. To study in more detail the early effects induced by AZT, the ability of the drug to interact with cardiolipin, an important component of internal mitochondrial membrane, was investigated by atomic force microscopy (AFM) in an artificial membrane model system. The results suggest that the primary effects of AZT may be related to a physical interference with the membrane structure leading to a consequent modification of its physical characteristics.

Lack of cross-resistance of a doxorubicin-resistant B16 melanoma line with 4'-deoxy-4'-iodo-doxorubicin.

A B16 melanoma cell line in which resistance to doxorubicin (Dx) had been induced by in vitro exposure to the drug, was found not to be cross-resistant with 4'-deoxy-4'-iodo-doxorubicin (4'-I-Dx), a new Dx derivative. Dx was 200 times less active in resistant than in sensitive cells, whereas the iodo derivative compound had the same level of activity in both cell lines. Cytotoxicity of Dx was dependent on concentration and on length of treatment, whereas that of 4'-I-Dx was correlated only with drug concentration. In an effort to explain this different behavior, intracellular retention and distribution of the two drugs was examined. Uptake and efflux of 4'-I-Dx in sensitive and resistant cells were similar, and cellular retention of the drug was 5-25 times higher than that of Dx. In addition, intracellular distribution of the iodo-derivative compound was similar in both cell lines, whereas more nuclear Dx was found in sensitive than in resistant cells. These differences may explain not only the lack of cross-resistance, but also the different cytotoxic behavior, of 4'-I-Dx.

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.

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.

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.

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.

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.