Leonarda Troiano

Multiparametric analysis of cells with different mitochondrial membrane potential during apoptosis by polychromatic flow cytometry

The analysis of changes in mitochondrial membrane potential (MMP) that can occur during apoptosis provides precious information on the mechanisms and pathways of cell death. For many years, the metachromatic fluorochrome JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide) was used for this purpose. Thanks to new dyes and to the technical improvements recently adopted in several flow cytometers, it is now possible to investigate, along with MMP, a variety of other parameters. Using three sources of excitation and polychromatic flow cytometry, we have developed a protocol that can be applied to cells undergoing apoptosis. In the model of U937 cells incubated with the chemopreventive agent quercetin (3,3′,4′,5,7-pentahydroxyflavone), we describe the detection at the single cell level of changes in MMP (by JC-1), early apoptosis (exposition of phosphatidylserine on the plasma membrane detected by annexin-V), late apoptosis and secondary necrosis (decreased DNA content by Hoechst 33342 and permeability of the plasma membrane to propidium iodide). The procedure can be completed in less than 2 h.

Inhibition of apoptosis by zinc: A reappraisal

Apoptosis--or programmed cell death--is an active type of cell death, occurring in several pathophysiological conditions. One of the most important characteristics of apoptosis is that cell death is preceded by DNA fragmentation, consequent to the activation of nuclear calcium- and magnesium-dependent endonuclease(s). DNA fragmentation can be inhibited by zinc ions. By using several techniques, such as DNA agarose gel electrophoresis, cytofluorimetric analysis of DNA content and of cell cycle, 3H-thymidine incorporation and trypan blue dye exclusion test, we show that zinc, despite completely inhibiting DNA fragmentation and the consequent loss of nuclear DNA content, does not protect rat thymocytes from spontaneous or dexamethasone-induced death. Our data also suggest that DNA fragmentation, although characteristic, is not a critical event for thymocyte death of apoptotic type.

Simultaneous analysis of reactive oxygen species and reduced glutathione content in living cells by polychromatic flow cytometry

Reactive oxygen species (ROS) are continuously produced in the cell as a consequence of aerobic metabolism, and are controlled by several antioxidant mechanisms. An accurate measurement of ROS is essential to evaluate the redox status of the cell, or the effects of molecules with the pro-oxidant or antioxidant activity. Here we report a cytofluorimetric technique for measuring simultaneously, at the single-cell level, hydrogen peroxide and superoxide anion, reduced glutathione (a main intracellular antioxidant) and cell viability. The staining is performed with the fluorescent dyes 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFH-DA), hydroethidine (HE), monobromobimane (MBB) and TO-PRO-3. This analysis is possible with new-generation flow cytometers equipped with several light sources (in our case, four lasers and an UV lamp), which excite different fluorochromes. This approach is extremely useful to study the balance between ROS content and antioxidants in cells receiving different stimuli, and to analyze the relationship between oxidative stress and cell death.

Characterization of Cells with Different Mitochondrial Membrane Potential During Apoptosis

Until now, the simultaneous analysis of several parameters during apoptosis, including DNA content and mitochondrial membrane potential (ΔΨ), has not been possible because of the spectral characteristics of the commonly used dyes. Using polychromatic flow cytometry based upon multiple laser and UV lamp excitation, we have characterized cells with different ΔΨ during apoptosis.

In vivo accumulation of sulfated glycoprotein 2 mRNA in rat thymocytes upon dexamethasone-induced cell death.

Two hours after a single intraperitoneal injection of dexamethasone (20 micrograms/Kg b.w.) into adult male rats, a typical ladder of DNA fragments was detectable upon separation on agarose gels of DNA from thymocytes. This became maximally evident at 4 hours. Accumulation of sulfated glycoprotein-2 (SGP-2) mRNA, whose rate of expression has been associated to the processes of programmed cell death, preceded the appearance of DNA degradation, starting to increase as early as 30 min after steroid injection, and maintained higher than controls until 8 hrs; a different time course was shown by changes in the levels of beta-actin mRNA. In the spleen, under the same conditions, the SGP-2 message also increased at 30 min, prior to DNA fragmentation, but decreased thereafter below the control value.

Thymic output and functionality of the IL‐7/IL‐7 receptor system in centenarians: implications for the neolymphogenesis at the limit of human life

During aging, the thymus undergoes a marked involution that is responsible for profound changes in the T‐cell compartment. To investigate the capacity of the thymus to produce new cells at the limit of human lifespan, we analyzed some basic mechanisms responsible for the renewal and maintenance of peripheral T lymphocytes in 44 centenarians. Thymic functionality was analyzed by the quantification of cells presenting the T‐cell receptor rearrangement excision circles (TREC). A new method based upon real‐time PCR was used, and we found that most centenarians (84%) had undetectable levels of TREC+ cells. Six‐color cytofluorimetric analysis revealed that centenarians had an extremely low number of naïve T cells; central memory and effector memory T cells were greatly increased, while terminally differentiated cells were as numerous as in young (aged 20–45) or middle‐aged (aged 58–62) donors. Interleukin (IL)‐7 and IL‐7 receptor α‐chain (CD127) levels were the same at all ages, as shown by ELISA, flow cytometry and real‐time PCR. However, IL‐7 plasma levels were higher in centenarian females than males. The presence of TREC+ cells and of very few naïve T lymphocytes suggests that in centenarians such cells could either derive from residues of thymic lymphopoietic islets, or even represent long‐living lymphocytes that have not yet encountered their antigen. IL‐7 could be one of the components responsible, among others, for the higher probability of reaching extreme ages typical of females.

Essential requirement of reduced glutathione (GSH) for the anti-oxidant effect of the flavonoid quercetin

We have analyzed the anti- or pro-oxidant effects of the flavonoid quercetin (QU) by evaluating, in U937 cell line, hydrogen peroxide (H2O2), superoxide anion reduced glutathione (GSH) content, mitochondrial membrane potential, DNA content, phosphatidylserine exposure on the outer face of the plasma membrane and cell viability. Polychromatic flow cytometry was used to evaluate in the same cells several functional parameters. For short periods of treatment QU exerted an anti-oxidant effect (decrease in H2O2 levels), whereas for long periods it showed a pro-oxidant activity (increase in ). In these conditions, GSH content was reduced, and this correlated with a lack of anti-oxidant activity of QU, which in turn could be correlated with proapoptotic activity of this molecule. Thus, QU can exert different effects (anti-/prooxidant) depending on exposure times and oxidative balance, and in particular on stores of GSH.

Genetic analysis of Paraoxonase (PON1) locus reveals an increased frequency of Arg192 allele in centenarians

Human Paraoxonase (PON1) is a High-Density Lipoprotein (HDL)-associated esterase that hydrolyses lipo-peroxides. PON1 has recently attracted attention as a protective factor against oxidative modification of LDL and may therefore play an important role in the prevention of the atherosclerotic process. Two polymorphisms have been extensively studied: a Leucine (L allele) to Methionine (M allele) substitution at codon 55, and a Glutamine (A allele) to Arginine (B allele) substitution at codon 192. We have examined these two aminoacidic changes in 579 people aged 20 to 65 years old, and 308 centenarians. We found that the percentage of carriers of the B allele at codon 192 (B+ individuals) is higher in centenarians than in controls (0.539 vs 0.447), moreover we found that among the B+ individuals, the phenomenon was due to an increase of people carrying M alleles at codon 55 locus. In conclusion, we propose that genetic variability at PON1 locus affects survival at extreme advanced age.

Mitochondrial heterogeneity during staurosporine-induced apoptosis in HL60 cells: analysis at the single cell and single organelle level.

BACKGROUND Apoptosis is a complex phenomenon during which several events occur. A growing interest exists on the role and functionality of mitochondria during this type of cell death. The responsibility of modifications in mitochondrial membrane potential (Delta Psi) in triggering apoptosis is under investigation. METHODS We evaluated Delta Psi changes in HL60 cells treated with staurosporine (STS). Flow cytometry and confocal microscopy have been used to analyze samples stained with two Delta Psi-sensitive probes, JC-1 and MitoTrackertrade mark Red CMXRos. RESULTS At the cellular level, we found heterogeneic behavior. Indeed, after STS treatment, some cells displayed typical markers of apoptosis and a collapse in Delta Psi. Others were apoptotic with no changes in Delta Psi, others changed Delta Psi without being apoptotic, and others were healthy. The same heterogeneic response to STS was found at the single organelle level. In a given cell, some mitochondria were depolarized whereas others were not. CONCLUSION In this model of apoptosis, changes in Delta Psi can be different among cells of the same type and among different organelles of the same cell. The collapse in Delta Psi is thus a heterogeneic phenomenon that seems to be an ancillary event following the irreversible phase of the apoptotic process.

Mitochondrial Functionality and Mitochondrial DNA Content in Lymphocytes of Vertically Infected Human Immunodeficiency Virus—Positive Children with Highly Active Antiretroviral Therapy—Related Lipodystrophy

Mitochondria functionality and apoptosis were studied in peripheral blood lymphocytes (PBL) of human immunodeficiency virus type 1-infected children, with or without lipodystrophy (LD), who were receiving highly active antiretroviral therapy (HAART) and in PBL of healthy control subjects (HCs). By flow cytometry, mitochondrial (mt) membrane potential, mt mass, intra-mt cardiolipin distribution, and early and late apoptosis in fresh PBL or in PBL cultured with different stimuli were assessed. mtDNA content was evaluated in fresh PBL by an original double-competitive quantitative polymerase chain reaction method, which enabled direct quantification of the number of mtDNA copies present in human lymphocytes. PBL from LD-positive and LD-negative children and from HCs were similar in mt functionality and in their tendency to undergo apoptosis. mtDNA content was also similar in PBL of LD-positive children and HCs, suggesting that normal mt functionality and normal tendency to undergo apoptosis are present in PBL of children with HAART-associated LD.