Daniela Barbieri

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.

Different pathways of apoptosis revealed by 2-chloro-adenosine and deoxy-D-ribose in mammalian astroglial cells

Both the adenosine analogue 2‐chloro‐adenosine (2‐CA) and the reducing sugar deoxy‐D‐ribose (dRib) induce apoptosis of astroglial cells in rat brain primary cultures (Abbracchio et al.: Biochem Biophys Res Commun 213:908–915, 1995). The present study was undertaken to elucidate by both morphological and cytofluorimetric analyses the intracellular mechanism(s) involved in induction of apoptosis by these two agents. The poly(ADP‐ribose)polymerase (PARP) inhibitor 3‐aminobenzamide did not prevent either 2‐CA‐ or dRib‐induced cell death, suggesting that activation of PARP is not critically important for induction of apoptosis in astrocytes. The radical scavenger N‐acetyl‐cysteine (NAC) strongly inhibited dRib‐ but not 2‐CA‐induced cell death, suggesting a differential role for radical formation in apoptosis by these two agents. A time‐dependent increase of cells with depolarized mitochondria was observed in dRib‐, and to a lesser extent, in 2‐CA‐treated cultures. NAC also prevented dRib‐ but not 2‐CA‐induced mitochondrial changes. We conclude that, in mammalian astrocytes, apoptosis can proceed through diverse and multiple pathways, depending upon the apoptotic stimulus. For dRib, apoptosis likely proceeds through generation of radicals and mitochondrial involvement. An adenosine extracellular receptor linked to an as yet unidentified signaling pathway may instead mediate 2‐CA‐induced cell death, which may have intriguing implications for both nervous system development and brain response to trauma and ischemia. J. Neurosci. Res. 47:372–383, 1997.

Apoptosis induced by 2-chloro-adenosine and 2-chloro-2'-deoxy-adenosine in a human astrocytoma cell line: differential mechanisms and possible clinical relevance.

We have previously demonstrated that 2‐chloro‐adenosine (2‐CA) can induce apoptosis of rat astroglial cells (Abbracchio et al. [1995] Biochem. Biophys. Res. Commun. 213:908–915). In the present study, we have characterized, for the first time, the effects induced on a human astrocytoma cell line (ADF cells) by both 2‐CA and its related analog 2‐chloro‐2′‐deoxy‐adenosine (2‐CdA, that is employed as anti‐cancer agent in chronic lymphoid malignancies). Exposure of these cells to either adenosine analog resulted in time‐ and concentration‐dependent apoptosis. Experiments with pharmacological agents known to interfere with adenosine receptors, its membrane transporter, and intracellular nucleoside kinases showed that: (i) cell death induced by either adenosine analog did not depend on extracellular adenosine receptors, but on a direct intracellular action; however, only in the case of 2‐CA, was entry into cells mediated by the specific nitrobenzyl‐tioinosine‐sensitive transporter; (ii) for both adenosine analogs, induction of apoptosis required the phosphorylation/activation by specific intracellular nucleoside kinases, i.e., adenosine kinase for 2‐CA, and deoxycytidine kinase for 2‐CdA. In addition, only in the case of 2‐CdA, was induction of apoptosis preceded by a block of cells at the G2/M phase of the cell cycle. Finally, at concentrations of either analog that killed about 80–90% of astrocytoma cells, a significantly lower effect on the viability of primary cortical neurons was observed. In conclusion, both adenosine analogs can trigger apoptosis of human astrocytoma cells, albeit with different mechanisms. This effect together with the relative sparing of neuronal cells, may have potential clinical implications for the therapy of tumors of glial origin. J. Neurosci. Res. 60:388–400, 2000

Adenosine A3 receptors and viability of astrocytes

We investigated the role of the A3 adenosine receptor in cells of the astroglial lineage (both rat primary astrocytes and human astrocytoma ADF cells) by means of the selective A3 agonists N6‐(3‐iodobenzyl)‐adenosine‐5′‐N‐methyluronamide (IB‐MECA) and CI‐IB‐MECA, and by utilizing the selective A3 receptor antagonist MRS1191. Exposure of ADF cells to μM concentrations of either agonist resulted in reduction of cell number, likely due to cell death. In both rat astrocytes and human astrocytoma cells, at concentrations 2–3 orders of magnitude lower (which were not associated with cytotoxicity), these same agonists induced a marked reorganization of the cytoskeleton, with appearance of stress fibers and numerous cell protrusions. Functionally, these morphological changes were associated with cell protection, as demonstrated by a significant reduction of spontaneous apoptosis in A3 agonist‐treated cells. To confirm a role for the A3 receptor in this effect, MRS1191 completely counteracted CI‐IB‐MECA‐induced reduction of spontaneous apoptosis. In ADF cells, A3 agonists also induced changes in the intracellular distribution of the anti‐apoptotic protein Bcl‐XL, which became localized in cell protrusions. Also, this effect was specifically antagonized by MRS1191. These dual actions of A3 agonists in vitro may have important in vivo implications. For example, a robust and acute activation of the A3 receptor following massive adenosine release during ischemia may contribute to brain cell death; conversely, a subthreshold activation of this receptor prior to ischemia may trigger protective mechanisms (i.e., induction of stress fibers and of a Bcl‐XL‐dependent reorganization of cytoskeleton) making the brain more resistant to subsequent insults (“ischemic tolerance”). Drug Dev. Res. 45:379–386, 1998.

Comparative and morphofunctional studies on mytilus galloprovincialis hemocytes: Presence of two aging‐related hemocyte stages

Abstract Morphological and functional studies were performed on hemocytes from young and adult Mytilus galloprovincialis. The results obtained suggest that only one cell type in two different stages, young or old, is present, consistently with the Mixs one‐cell‐type model. However, both young and old hemocytes appear capable of carrying out similar functions and express common signal molecules; thus, their differences in cytology and number appear to be due to cellular aging.

Modulation of cis-diamminedichloroplatinum (II) accumulation and cytotoxicity by spermine in sensitive and resistant human ovarian carcinoma cells

The effect of spermine (Sp), a natural polycationic amine, on cisplatin (CDDP) sensitivity and accumulation of a human ovarian CDDP-sensitive cell line (2008) and its resistant variant (C13*) was investigated. Survival was also studied. The C13* cells were approximately 20-fold resistant to CDDP, yet were found to be just as sensitive to Sp as 2008 cells. When Sp was concurrently added with CDDP to the colony-forming assay, the IC50 dose was approximately 3-fold lower than that of CDDP alone. This decrease was the result of a synergistic interaction, as assessed by median effect analysis. The incubation of cells with the approximate IC50 dose of Sp for 1-8 h indicated that this synergism could be due to stimulation of CDDP accumulation, showing maximal uptake after 4 h of Sp exposure. This stimulation may be the result of a modulation of cellular membrane permeability by Sp, as assessed by the accumulation of [3H]mannitol. Exposure to Sp concentrations active on CDDP uptake also significantly increased [3H]mannitol accumulation in both cell lines. The triamine spermidine (Spd) did not significantly affect either the sensitivity of the two cell lines or CDDP and [3H]mannitol accumulation. These results suggest that Sp is a positive modulator of CDDP uptake, and thus of its cytotoxicity, even in resistant cells, where the phenotype is partly due to a CDDP accumulation defect.

Cell Proliferation and Cell Death in Immunosenescence

Aging is characterized by a variety of alterations that occur in most organs and cell types. Loss of proliferative vigor is considered a marker of the aging process and is related to the Hayflick phenomenon, that is, the limited number of replications that normal cells can undergo. A correlation between proliferative capability and maximum lifespan in different species, and an inverse correlation between proliferative capability and donor age have been dem~nstrated.l-~ Thus, a decreased capability to proliferate may be considered a characteristic of cellular senescence. In a recent paper we argued that an intriguing relation exists among cellular senescence, tumor growth, and 10ngevity.~ All these phenomena are deeply related to programmed cell death or apopto~is.~ A possible scenario is the following: cells may be equipped with genes that actively promote cellular senescence, thus controlling cell death in order and escaping transformation.68 This situation is balanced by other genes responsible for survival and viability, as depicted in FIGURE 1. Circumstantial evidence suggests that cellular senescence may be considered a peculiar type of cell differentiation whose biological function is to counteract uncontrolled cell proliferation. (For an example, see ref. 9.) From this point of view, cellular senescence can be considered one of the most important mechanisms in avoiding the continuous onset of tumors. The most effective evolutive way for a cell to control neoplastic growth is likely to set up genes that promote apoptotic cell death.1s24

Factors predicting the outcome of conservatively treated adenocarcinoma in situ of the uterine cervix: An analysis of 166 cases

OBJECTIVE The present study assessed the clinical outcome of patients conservatively treated for cervical adenocarcinoma in situ (AIS) and their predictive factors using univariate and multivariate population averaged (PA) generalized estimating equation (GEE) model in a longitudinal setting. METHODS A series of 166 consecutive women (mean age 39.8 yrs; range 23-63 yrs) underwent conservative treatment of AIS as the primary treatment and were followed-up (mean 40.9 mo) using colposcopy, PAP-smear, biopsy and HPV-testing with Hybrid Capture 2. RESULTS Hysterectomy was performed as part of the primary management in 47 patients, who were excluded from the follow-up (FU) analysis. Out of 119 women closely followed-up, additional therapeutic procedures were performed in 69. At study conclusion, 7 patients (5.9%) showed persistent disease, while 8 (6.7%) had progressed to invasive adenocarcinoma (AC). Positive HR-HPV test was the only independent predictor of disease recurrence (adjusted OR=2.72; 95%CI 1.08-6.87), and together with free cone margins (OR=0.20; 95%CI 0.04-0.92), HR-HPV positivity was also the single most powerful predictor of disease progression to AC, with OR=3.74; 95%CI 1.84-7.61 (p=0.0001) in multivariate PA-GEE. CONCLUSIONS These results suggest that testing HR-HPV positive at any time point during FU is the most significant independent predictor of progressive disease, while showing free margins in cone has a significant protective effect against progression to AC. Furthermore, because 4.3% women with persistent, recurrent or progressive disease experienced a late (5th and 6th FU) diagnosis of HG-CGIN or microinvasive AC, a close surveillance should be scheduled for at least three years in conservatively treated AIS patients.

N1,N12-bis(ethyl)spermine effect on growth of cis-diamminedichloroplatinum(II)-sensitive and -resistant human ovarian-carcinoma cell lines

The results presented here demonstrate that a cis‐diamminedichloroplatinum(II) (DDP)‐resistant human ovarian‐carcinoma cell line is also cross‐resistant to the spermine analogue N1,N12‐bis(ethyl)spermine (BESPM). We report that C13* cells, which are approximately 20‐fold resistant to DDP, similarly showed 7‐fold resistance to BESPM by colony‐forming assay with an IC50 value of 24.6 ± 2 μM vs. 3.4 ± 0.8 μM of 2008 cells. Resistance appears to be the result of many effects, such as different morphological and functional modifications of mitochondria. Furthermore, although BESPM accumulation was almost identical in sensitive and resistant cells, the intracellular polyamine pool of the 2 cell lines was differentially affected by this polyamine analogue. In fact, when spermidine (SPD) was still detectable in C13* cells, in 2008 cells it was not, and the spermine (SPM) content was always more markedly reduced in sensitive cells than in the resistant variant. The lower polyamine content of 2008 cells could be related to a higher degree of induction of spermidine/spermine N1‐acetyltransferase (SSAT) activity by BESPM in sensitive cells than in their resistant counterpart. Despite the observed cross‐resistance, the combination of the 2 drugs resulted in supra‐additive and synergistic effects in both cell lines, depending on concentration, as assessed by median‐effect analysis of the survival data. The effectiveness of this combination was also confirmed by the increased accumulation of cells in the G2/M phase of the cell cycle in both cell lines. Taken together, these data suggest that BESPM effect on cell growth of DDP‐sensitive and DDP‐resistant cells involves multiple mechanisms that are differently modulated by the DDP‐resistant phenotype. Int. J. Cancer 78:33–40, 1998.© 1998 Wiley‐Liss, Inc.

Neutral endopeptidase-24.11 (NEP) activity in human fibroblasts during development and ageing

Neutral endopeptidase-24.11 (NEP, EC 3.4.24.11) is a cell surface Zn metallopeptidase that hydrolyzes bioactive regulatory peptides. Using a spectrofluorimetric procedure, we assessed NEP activity in plasma membranes of normal human skin and lung fibroblasts. We found a considerable increase in NEP activity during fetal-to-adult transition. Adult skin fibroblasts from an old donor exhibited significantly higher levels of NEP activity than cells from young donors. Interestingly, however, the NEP activity of fibroblasts from a centenarian donor was similar to that of cells from young donors. Increased levels of NEP activity were also found in in vitro aged lung fibroblasts. Finally, adrenocorticotropin hormone (ACTH (1-24)), a regulatory peptide that can be cleaved by NEP, provoked an increase in enzymic activity in fetal and young adult donor fibroblasts and a decrease in this activity in fibroblasts from adult and old donors. This finding suggests that ageing may affect NEP activity.