Roberta Ferraresi

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.

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.

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.

Mitochondrial alterations and tendency to apoptosis in peripheral blood cells from children with Down syndrome

Different types of cells from subjects with Down syndrome (DS) have an increased susceptibility to cell death. We have studied apoptosis and mitochondrial (mt) membrane potential (ΔΨ m) in peripheral blood mononuclear cells (PBMC) from DS children and age‐matched healthy donors after in vitro treatment with apoptogenic molecules, along with mtDNA content. We found that PBMC from DS and healthy controls had a similar tendency to undergo apoptosis and a similar amount of mtDNA. However, in cells from DS subjects, mitochondria showed a higher loss of ΔΨ m, underlying the presence of an increasing susceptibility of these organelles to damaging agents.

Homeostatic cytokines and expansion of regulatory T cells accompany thymic impairment in children with Down syndrome.

Down syndrome (DS), the most common chromosomal abnormality in humans, is characterized by precocious immunologic aging that results, among other things, in alterations of B and T lymphocyte subsets and natural killer cells, defective phagocytosis, and chemotaxis of polymorphonuclear leukocytes. We studied 30 children affected by DS, compared them to 29 healthy controls, and evaluated the functionality of the thymus (by measuring the amount of lymphocytes that express the signal-joint T cell receptor rearrangement excision circles [sj-TREC+]), the plasma levels of interleukin (IL)-7 and IL-15, the proliferative T cell response to these cytokines, the expression of the alpha chain of the IL-7 receptor (CD127), the extrathymic differentiation of T lymphocytes, and the presence of natural regulatory T cells (Tregs) in peripheral blood. We found that DS children had a significantly lower number of sj-TREC+ lymphocytes, the levels of which were strongly correlated with age. We found higher plasma levels of IL-7 and IL-15 than in healthy controls, and a higher proliferative T cell response to IL-15. DS children also showed a lower percentage of CD4(+) cells and profound alterations of T cell differentiation, along with increased amount of Tregs and of cells expressing markers of apoptosis. We can thus hypothesize that the precocious thymic involution occurring in DS is mirrored by a high production of IL-7 and IL-15, which is crucial for cell survival and proliferation. The complex alterations present in the periphery are likely the result of a compensatory mechanism: the overproduction of homeostatic cytokines could be a reaction to the impaired intrathymic production of T lymphocytes and/or to the expansion of Treg in the periphery, and could be required to allow the survival of T cells.

Resistance of mtDNA-depleted cells to apoptosis

Cells lacking mitochondrial genome (defined as ρ0) are useful models in studies on cancer, aging, mitochondrial diseases and apoptosis, but several of their functional aspects have been poorly characterized. Using different clones of ρ0 cells derived from the human osteosarcoma line 143B, we have tested the effects of different apoptogenic molecules such as staurosporine (STS), doxorubicin, daunomycin and quercetin, and have analyzed apoptosis, mitochondrial membrane potential (MMP), levels of oxygen free radicals, reduced glutathione (GSH) content, and expression of P‐glycoprotein (P‐gp). When compared to parental cells, ρ0 cells resulted much less sensitive to apoptosis. MMP was well maintained in ρ0 cells, and remained unchanged after adding apoptogenic agents, and did not change after treatment with molecules able to depolarize mitochondria such as valinomycin. After adding STS, the production of reactive oxygen species was similar in both cell types, but ρ0 cells maintained higher levels of GSH. In ρ0 cells, P‐gp was strongly over‐expressed both at mRNA and protein level, and its functionality was higher. The resistance to apoptosis of ρ0 cells could be not only due to an increased scavenger capacity of GSH, but also due to a selection of multidrug resistant cells that hyperexpress P‐gp.

HOXD13 Binds DNA Replication Origins To Promote Origin Licensing and Is Inhibited by Geminin

ABSTRACT HOX DNA-binding proteins control patterning during development by regulating processes such as cell aggregation and proliferation. Recently, a possible involvement of HOX proteins in replication origin activity was suggested by results showing that a number of HOX proteins interact with the DNA replication licensing regulator geminin and bind a characterized human origin of replication. The functional significance of these observations, however, remained unclear. We show that HOXD13, HOXD11, and HOXA13 bind in vivo all characterized human replication origins tested. We furthermore show that HOXD13 interacts with the CDC6 loading factor, promotes pre-replication complex (pre-RC) proteins assembly at origins, and stimulates DNA synthesis in an in vivo replication assay. HOXD13 expression in cultured cells accelerates DNA synthesis initiation in correlation with the earlier pre-RC recruitment onto origins during G1 phase. Geminin, which interacts with HOXD13 as well, blocks HOXD13-mediated assembly of pre-RC proteins and inhibits HOXD13-induced DNA replication. Our results uncover a function for Hox proteins in the regulation of replication origin activity and reveal an unforeseen role for the inhibition of HOX protein activity by geminin in the context of replication origin licensing.

Protective effect of acetyl-L-carnitine against oxidative stress induced by antiretroviral drugs.

Both HIV infection per se and antiretroviral drugs might contribute to oxidative stress and mitochondrial dysfunctions. In this study we assess zidovudine, stavudine and didanosine on U937 and CEM cell lines. All these drugs induced apoptosis and increased intracellular hydrogen peroxide but not superoxide anions. The addition of acetyl‐l‐carnitine (ALC) was able to prevent the pro‐oxidant effect of the drugs tested. Supplementation with ALC, deficient in certain cohorts of HIV‐infected individuals, especially on high active antiretroviral therapy regimen, has been associated with favourable effects. These data suggest that one of these effects could be a direct anti‐oxidant action.