Cellular senescence can occur with similar phenotypes in normal cells, during aging, and in tumor cells, spontaneously or after cytostasis. The fall or increase in proliferative activity are key aspects of the respective conditions, in which the levels of reactive oxygen species can vary, affecting the cellular redox homeostasis. This work aimed to study the relationships between senescence and transformation by comparing cells with different proliferative activities and phenotypes attributable to transformation (NIHs cultures) or senescence (NIHv cultures), before and after incubation with hydrogen peroxide. Both cultures were derived from the NIH/3T3 cell line, which was used here as a reference (NIHb), after the serum starvation. Our experimental model can be representative of the heterogeneity of cell subpopulations, with different degrees of transformation and senescence, found in some tumors. The characterization of the functional properties of NIHb, NIHs, and NIHv cells was performed by a morphocytometric analysis of the cell cycle progression, mitochondrial and lysosomal content/activity, and superoxide anion production. The efficiency of the lysosomal compartment was also assessed by estimating the autophagic activity and measuring lipofuscin autofluorescence. Comparisons of nuclear and cytoplasmic parameters before and after the incubation with hydrogen peroxide revealed differences in the expression and modulation of cellular senescence patterns. The treatment effects were very limited in the NIHb culture; the senescence condition was essentially maintained in the NIHv cells, while the most relevant changes were found in the NIHs cells. In the latter, the acquisition of the senescent phenotype, also demonstrated by the positivity of SA-β-galactosidase, was correlated with a decrease in proliferative activity and a change in the content/activity of the mitochondria and lysosomes, which showed similarities with the basal senescence conditions of NIHv cells. In NIHs cells, increased autophagy events and lipofuscin accumulation also indicate the establishment of cytoplasmic dynamics typical of senescence. The variable responses to hydrogen peroxide, besides depending on the different basal cytokinetic activity of the cultures examined, appeared to be related to the specific cell redox state resulting from the balance between endogenous ROS and those produced after treatment. Especially in NIHs cells, the slowing down of the cell cycle was linked to dynamic interconnections between the mitochondrial and lysosomal compartments. This would indicate that transformed cells, such as NIHs, may express morpho-functional aspects and markers typical of cellular senescence, as a consequence of the modulation of their redox state.

Background Neoplastic subpopulations can include polyploid cells that can be involved in tumor evolution and recurrence. Their origin can be traced back to the tumor microenvironment or chemotherapeutic treatment, which can alter cell division or favor cell fusion, generating multinucleated cells. Their progeny, frequently genetically unstable, can result in new aggressive and more resistant to chemotherapy subpopulations. In our work, we used NIHs cells, previously derived from the NIH/3T3 line after serum deprivation, that induced a polyploidization increase with the appearance of cells with DNA content ranging from 4 to 24c. This study aimed to analyze the cellular dynamics of NIHs culture subpopulations before and after treatment with the fusogenic agent polyethylene glycol (PEG), which allowed us to obtain new giant polyploid cells. Successively, PEG-untreated and PEG-treated cultures were incubated with the antimicrotubular poison vinblastine. The dynamics of appearance, decrease and loss of cell subpopulations were evaluated by correlating cell DNA content to mono-multinuclearity resulting from cell fusion and division process alteration and to the peculiarities of cell death events. Results DNA microfluorimetry and morphological techniques (phase contrast, fluorescence and TEM microscopies) indicated that PEG treatment induced a 4–24c cell increase and the appearance of new giant elements (64–140c DNA content). Ultrastructural analysis and autophagosomal–lysosomal compartment fluorochromization, which allowed us to correlate cytoplasmic changes to death events, indicated that cell depletion occurred through distinct mechanisms: apoptotic death involved 2c, 4c and 8c cells, while autophagic-like death involved intermediate 12–24c cells, showing nuclear (lobulation/micronucleation) and autophagic cytoplasm alterations. Death, spontaneously occurring, especially in intermediate-sized cells, was increased after vinblastine treatment. No evident cell loss by death events was detected in the 64–140c range. Conclusions PEG-treated NIHs cultures can represent a model of heterogeneous subpopulations originating from cell fusion and division process anomalies. Altogether, our results suggest that the different cell dynamics of NIHs subpopulations can affect the variability of responses to stimuli able to induce cell degeneration and death. Apoptptic, autophagic or hybrid forms of cell death can also depend on the DNA content and ability to progress through the cell cycle, which may influence the persistence and fate of polyploid cell descendants, also concerning chemotherapeutic agent action.

OBJECTIVES:To analyse proliferation, differentiation and apoptosis in THP-1 cells after stimulation with phorbol 12-myristate 13-acetate (PMA) and retinoic acid (RA).MATERIALS AND METHODS:PMA and RA were used in a three-step-procedure: (i) treatment with 6, 30, 60 nm PMA, that induced initial, intermediate and advanced levels of monocyte-macrophage transition, respectively; (ii) recovery in PMA-free medium; (iii) incubation with 4 μm RA. Cultures were characterized cytokinetically (flow cytometry/bromodeoxyuridine uptake) and immunocytochemically (static cytometry) for expression of CD14, CD11b (monocyte-macrophage) and DC-SIGN (dendritic cell: DCs) markers.RESULTS:Some treatments determined appearance of monocyte/macrophage, dendritic and apoptotic phenotypes, percentages of which were related to PMA dose used in step 1, and dependent on presence/absence of PMA and RA. PMA withdrawal induced dedifferentiation and partial restoration of proliferative activity, specially in 6 and 30 nm PMA-derived cells. Recovery in the presence of serum (fundamental to DC appearance) indicated that depending on differentiation level, cell proliferation and apoptosis were inversely correlated. Treatment with 30 nm PMA induced intermediate levels of monocytic-macrophagic differentiation, with expression of alternative means of differentiation and acquisition of DCs without using cytokines, after PMA withdrawal and RA stimulation.CONCLUSIONS:Our experimental conditions favoured differentiation, dedifferentiation and transdifferentiational pathways, in monocytic THP-1 cells, the balance of which could be related to both cell proliferation and cell death.

In this work, the influence of different substrate adhesion during phorbol-12-myristate-13-acetate (PMA)induced differentiation of THP-1 monocytic cell line was studied.In particular, by morphocytochemical and cytometric approaches, the influence of type I and type IV collagens in an experimental model representative of three phases (initial, intermediate and terminal) of monocyte-macrophage transition was analyzed.The cells in these three phases of differentiation were obtained by using 6, 30 e 60 nM PMA.In this experimental model, referring to adhesion to glass as control, by using the azo-dye coupling method, we have considered the analysis of Acid Phosphatase (AcP) activity as a marker of differentiated status expression, in relation to the acquisition of macrophagic phenotype.Endosomal/lysosomal system was further characterized by taking into account the uptake of fluorescent probe LysoTracker Red.Fluorochromization in the various experimental conditions was analyzed morphologically (fluorescence microscopy) and quantitatively (static cytometry).Data related to lysosome compartment were integrated, from a cytokinetic point of view, by flow cytometry measurements of DNA/protein content.Our results have indicated that type I and type IV collagens were able to influence, with respect to glass adhesion, various differentiation phases.Type I collagen showed the higher effects in the condition of high differentiation (60 nM PMA), causing an increase in AcP activity and lysosomal system.Type IV collagen, besides determining effects on lysosomal compartment of intermediate and terminally differentiated cells, influenced mainly proliferative activity of cells with initial differentiation level (6 nM PMA).

The effects induced by different concentrations (50, 75, 100 microM) of the cytostatic drug cisplatin (cDDP) in NIH/3T3 cells were analyzed. Sub-confluent cultures of this mouse fibroblast line, obtained after serum deprivation, showed the presence of aneuploid/polyploid cells with ploidy values ranging from 4c to 24c. DNA content cytofluorometry demonstrated that 50 and 75 microM cDDP induced a cytostatic effect; 100 microM concentration showed lower antiproliferative action. All treatments caused a partial cell detachment and apoptosis, the incidence of which appeared to be cDDP concentration-dependent. Ultrastructural and fluorescence microscopy integrated analyses of the still adherent cells demonstrated the presence of alternative degeneration patterns, especially in polyploid cells, with extensive modifications at both nuclear and cytoplasmic levels. There were events of micronucleation and phenomena of multilobulation and furrows of the nucleus that preceded the formation of heterogeneous fragments. These events were correlated, at cytoplasmic level, with actin reorganization and the appearance of autophagocytotic processes. In our cell model, the same pharmacological treatment was able to induce different cell death phenomena relating to cell dimension and ploidy. More actively proliferating cells (2c-4c DNA content) die throughout canonical apoptosis, while polyploid cells prevailingly degenerate by mechanisms partly referable to autophagic cell death.

The changes of the microtubular network induced by microtubule destabilizing (Vinblastine, VBL) and stabilizing (Taxol, TAX) agents were studied in NIH/3T3 fibroblastic cells in conventional culture conditions (NIHb) and in a subpopulation (NIHs), obtained after serum deprivation and expressing different morphofunctional features and higher cytokinetic activity. In this cell model, we analyzed VBL and TAX effects on cell cycle and microtubular network, in relation to cell death. In NIHb cells, VBL induced higher microtubule depolymerization, prevalence of tubulin paracrystals and micronucleation, while, in NIHs cells, lower depolymerization and appearance of tubulin spiral-like structures, with lower micronucleation, increase of apoptosis and disappearance of high polyploid cells. DNA static cytofluorometry of cells showing paracrystals or spirals permitted correlation of the appearance of these tubulin aggregation forms with the cell cycle phases. In NIHb cultures, the DNA content curves, in cells with paracrystals or spirals, showed a similar trend, with a higher frequency of the two anomalies in the G2/M phase. In NIHs cultures, paracrystals and spirals are found in G2/M cells, while G1 cells showed prevailingly paracrystals. TAX induced the appearance of microtubule bundles in the two cultures. The prevalence of circular bundles was found in NIHb cells, while a higher number of linear bundles was shown in NIHs cells. In NIHb cells, circular bundles were related to higher apoptosis and micronucleation. DNA cytofluorometry, in cells with linear or circular bundles, showed that the latter was present with high frequency in NIHb cells in all the cell cycle phases; in NIHs cells, they appeared, with lower frequency, prevailing in the S-G2/M phase. Furthermore, in NIHs cells, the appearance of linear bundles in G1 cells was related to a lower micronucleation. These finding showed that microtubule reorganization in different cell cycle phases could play a role in the progression of nuclear fragmentation/micronucleation relating to cell death.

The different effects of two cytostatic drugs, Actinomycin D and Vinblastine, during macrophage-like differentiation induced in THP-1 monocytic cell line by phorbol ester phorbol 12-myristate 13-acetate (PMA) (6, 30, and 60 nM), were studied by morpho-cytochemical approaches. In PMA-unstimulated monocytic cells, the cytostatic effects of Actinomycin D (an antimetabolic drug) were characterized by a drastic reduction of the G2/M cells accompanied by dramatic death of the G1 cells; on the contrary, Vinblastine (a microtubule-depolymerizating drug) induced an accumulation of the G2/M cells with the appearance of aneugenic micronuclei and scarce cell death mainly from the G1 cells. After 60 nM PMA stimulation, the culture was mostly composed by macrophagic cells characterized by low proliferation and the appearance of mono-/binucleated polyploid cells; in this condition, the cytotoxicity of the two drugs, more effective for Vinblastine, induced cell death in the different ploidy classes (2c, 4c, 8c). Cell death appeared to be of apoptotic nature, but with some morpho-phenotypic differences due to the action mechanism of the drugs and dependent on cell culture growth and differentiation. As a consequence of the different block-action of the two drugs on the cell cycle phases and in relation to the different subcellular targets, the effects changed during the transition from not-adhering/proliferating monocytes to adhering/low-proliferating differentiated macrophages.

The relationship between apoptotic progression and cell cycle perturbation induced by microtubule-destabilising (vinblastine, Colcemid) and -stabilising (taxol) drugs was studied in two mesenchyme-derived neoplastic cell lines, growing as suspension (Jurkat) and monolayer (SGS/3A) culture, by morphocytochemical and biochemical approaches. The same kind of drug induced different effects on the cell kinetics (proliferation, polyploidisation, death) of the two cell lines. In floating cells, the drugs appeared more effective during the S phase, while in adherent cells they were more effective during the G2/M phase. Moreover two distinct neoplasia-associated apoptotic phenotypes emerged: the first pattern was the typical one and was found in cells with a low transition through the S/G2 phase (Jurkat), and the second one was mainly characterised by a cell death derived from micronucleated and mitotic cells, as a consequence of a low transition through the M/G1 phase (SGS/3A). Our data show that the machinery required for the trigger and progression of apoptosis is present in every cell cycle phase, also in conditions of karyological alterations (aneugenic micronucleations). On the other hand, a different sensitivity of the two microtubular components (interphasic network and mitotic spindle) appears to be related to the anchorage-dependence or -independence during the cell growth disturbances after exposure to antimicrotubular drugs.

Swiss mice were given 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 25 mg/kg/day, for 5 consecutive days and killed at different days after MPTP discontinuance. Decreases in striatal tyrosine hydroxylase activity and levels of dopamine and its metabolites were observed I day after MPTP discontinuance. Ascorbic acid and glutamate levels had increased, dehydroascorbic acid and GSH decreased, whereas catabolites of high-energy phosphates (inosine, hypoxanthine, xanthine, and uric acid) were unchanged. In addition, gliosis was observed in both striatum and substantia nigra compacta SNc. Sections of SNc showed some terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling (TUNEL)-positive cells. Neurochemical parameters of dopaminergic activity showed a trend toward recovery 3 days after MPTP discontinuance. At this time point, TUNEL-positive cells were detected in SNc; some of them showed nuclei with neuronal morphology. A late (days 6-11) increase in striatal dopamine oxidative metabolism, ascorbic acid oxidative status, and catabolites of high-energy phosphates were observed concomitant with nigral neuron and nigrostriatal glial cell apoptotic death, as revealed by TUNEL, acridine orange, and Hoechst staining, and transmission electron microscopy. These data suggest that MPTP-induced activation/apoptotic death of glial cells plays a key role in the sequential linkage of neurochemical and cellular events leading to dopaminergic nigral neuron apoptotic death.

BACKGROUND:Microvascular insufficiency represents a major cause of end-organ failure among diabetics.METHODS AND RESULTS:In streptozotocin-induced diabetic mice, we evaluated the potential of human tissue kallikrein (hTK) gene as a sole therapy against peripheral microangiopathy. Local delivery of hTK gene halted the progression of microvascular rarefaction in hindlimb skeletal muscle by inhibiting apoptosis, thus ensuring an improved hemodynamic recovery in case of supervening vascular occlusion. The curative action of hTK did not necessitate insulin supplementation. Application of gene therapy at a stage of established microangiopathy stimulated vascular regeneration.CONCLUSIONS:Our studies indicate that hTK may represent a useful tool for the treatment of microvascular complications in diabetics.

In this study we investigated the relationship between the reorganisation of actin cytoskeleton and the changes at cell surface level (i.e. PS exposure and blebbing) in two neoplastic cell lines during apoptosis: Chang liver cells (adherent culture) and promyelocytic HL-60 cells (suspension culture), treated with the podophyllotoxin derivative VP16. The morphological analysis, performed by means of conventional fluorescence microscopy and confocal laser scanning microscopy, on Chang cells showed that onset and progress of the two processes are synchronised. The initial disassembly of stress fibers was associated with the early PS exposure on the cell surface. Moreover, the accumulation of actin at cortical level appeared strongly associated with an intense labelling for Annexin V and, in some cases, especially in the areas of membrane blebbing. The double staining for actin and PS exposure, quantitatively analysed by flow cytometry in HL-60 cells after different treatment times, demonstrated that the decrease of Annexin V binding in the late stages of apoptosis is associated with the strong reduction of actin labelling probably also due to a proteolytic cleavage. These events were also partially related to variations of the functional state of mitochondria, by analysing cytofluorometrically the dissipation of the inner membrane potential (delta psi m).

Get PDF Email Share Share with Facebook Tweet This Post on reddit Share with LinkedIn Add to CiteULike Add to Mendeley Add to BibSonomy Get Citation Copy Citation Text A. C. Croce, A. Spano, S. K. Lanza, D. Locatelli, S. Barni, L. Sciola, and G. Bottiroli, "Autofluorescence in single living cells: a parameter for in situ, real time monitoring of metabolism," in Biomedical Optical Spectroscopy and Diagnostics, T. Li, ed., Vol. 38 of OSA Trends in Optics and Photonics (Optica Publishing Group, 2000), paper MC4. Export Citation BibTex Endnote (RIS) HTML Plain Text Citation alert Save article

In the present study we analysed the changes in cytoskeleton actin in lymphoid cells following IL-2 activation and during cell interactions by means of light and electron microscopy, immunofluorescence and molecular analysis. By morphological analysis we observed a higher fluorescence in the activated cells than in the quiescent ones with no modifications in the cytoskeleton pattern comparing activated to resting cells. The results of molecular analysis indicate that, after IL-2 activation, there is a reorganisation of the actin component of the cell cytoskeleton accompanied by the differential expression of the corresponding genes. A future study will be extended to the analysis of others components of the cytoskeleton network.

Abstract— The dependence of autofluorescence properties on the metabolic and functional engagement and on the transformation condition was studied on single cells. Normal Galliera rat fibroblasts at low subculture passage (cell strain), at high subculture passage (stabilized cell line), and transformed ceil line derived from a rat sarcoma were used as a cell model. The study was performed by microspectrofluorometric and fluorescence imaging technique. The autofluorescence properties of cells were studied by excitation at two wavelengths, namely 366 nm and 436 nm, that are known to favor the emission of different fluorophores. Spectral shape analysis indicated that under excitation at 366 nm autofluorescence is ascribable mainly to coenzyme molecules, particularly to reduced pyridine nucleotides, while under excitation at 436 nm, flavin and lipopigment emission is favored. The energetic metabolic engagement of the different cell lines was analyzed in terms both of parameters related to anaerobicaerobic pathways (biochemical assay) and of mitochondrial features (supravital cytometry). The results showed that the cell strain and the stabilized and transformed cell lines can be distinguished from one another on the basis of both overall fluorescence intensity and the relative contributions of spectral components. These findings indicated a relationship between autofluorescence properties and energetic metabolism engagement of the cells that, in turn, is dependent on the proliferative activity and the transformed condition of the cells. In that it is a direct expression of the energetic metabolic engagement, autofluorescence can be assumed as an intrinsic parameter of the cell biological condition, suitable for diagnostic purposes.

Abstract : L‐DOPA and manganese both induce oxidative stress‐mediated apoptosis in catecholaminergic PC12 cells. In this study, exposure of PC12 cells to 0.2 mM MnCl2 or 10‐20 μM L‐DOPA neither affected cell viability, determined by the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay, nor induced apoptosis, tested by flow cytometry, fluorescence microscopy, and the TUNEL technique. L‐DOPA (50 μM) induced decreases in both cell viability and apoptosis. When 0.2 mM MnCl2 was associated with 10, 20, or 50 μM L‐DOPA, a concentration‐dependent decrease in cell viability was observed. Apoptotic cell death also occurred. In addition, manganese inhibited L‐DOPA effects on dopamine (DA) metabolism (i.e., increases in DA and its acidic metabolite levels in both cell lysate and incubation medium). The antioxidant N‐acetyl‐L‐cysteine significantly inhibited decreases in cell viability, apoptosis, and changes in DA metabolism induced by the manganese association with L‐DOPA. An increase in autoxidation of L‐DOPA and of newly formed DA is suggested as a mechanism of manganese action. These data show that agents that induce oxidative stress‐mediated apoptosis in catecholaminergic cells may act synergistically.

Reportedly, the generation of nitric oxide (NO) may lead to iron mobilization from ferritin disrupting intracellular iron homeostasis and increasing levels of reactive oxygen species. In the present study, we evaluated the role of endogenous iron in NO-induced apoptosis in PC12 cells. Apoptosis was tested by flow cytometry, fluorescence microscopy and terminal deoxynucleotidyl transferase-mediated 2'-deoxy-uridine 5'-triphosphate nick end labeling (TUNEL) technique. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. When incubated with 0.5-0.75 mM sodium nitroprusside (SNP, a chemical NO donor), PC12 cells were shown to undergo apoptosis. In addition, SNP induced a time-dependent decrease in cell viability. Since deferoxamine (0.05-0.1 mM), a powerful iron chelator, inhibited both SNP-induced apoptosis and the decrease in cell viability, we suggest that these NO effects may be dependent upon iron mobilization within the cell.