C. Pellicciari

Estrogen and progesterone induction of survival of monoblastoid cells undergoing TNF-α-induced apoptosis

Induction of apoptosis of mononucleated cells is a physiological process for regulating the intensity of the immune response. The female steroid hormones estrogen (E2) and progesterone (Prog) are known to modulate the reactivity of the immune system; recently it has been demonstrated that they can regulate induction of apoptosis of endothelial cells and osteoblasts. TNF‐α‐mediated induction of apoptosis has been well characterized in myeloid cells. We investigated whether E2 and Prog could interfere with TNF‐α‐induced apoptosis of the monoblastoid U937 cell line. Treatment with E2 or Prog increased survival and prevented apoptosis induced by TNF‐α in both undifferentiated and macrophage‐like PMA‐differentiated U937 cells, as assessed by trypan blue exclusion cell counting, thymidine incorporation, AnnexinV labeling, followed by flow cytometry and DNA fragmentation studies. This effect can be associated with the activation of specific hormone receptors, since we observed the expression of the estrogen receptor α (ER‐α), ER‐β, and progesterone receptor (PR) mRNAs; the ER‐α protein expression was confirmed by immunocytochemical analysis. In addition, hormone‐mediated survival against apoptosis was concentration dependent, reaching the half‐maximal effect at 10 nM and blocked by the ER antagonist ICI 182,780 in undifferentiated cells, further supporting a receptor‐mediated mechanism of cell survival. Other steroid receptor drugs such as Raloxifene, RU486, or the ICI 182,780 in PMA‐differentiated cells displayed agonist activity by preventing TNF‐α‐induced apoptosis as efficiently as the hormones alone, providing further evidence to the notion that steroid receptor drugs may manifest agonist or antagonist activities depending on the cellular context in which they are studied. Treatment with E2 was also associated with a time‐dependent decrease in the mRNA level of the proapoptotic Nip‐2 protein, supporting the hypothesis that hormone responsiveness of U937 cells is mediated by target gene transcription. Together, these results demonstrate that ER and PR can be activated by endogenous or exogenous ligands to induce a genetic response that impairs TNF‐α‐induced apoptosis in U937 cells. The data presented here suggest that the female steroid receptors play a role in regulation of the immune response by preventing apoptosis of monoblastoid cells; this effect might have important consequences in the clinical use of steroid receptor drugs.—Vegeto, E., Pollio, G., Pellicciari, C., Maggi, A. Estrogen and progesterone induction of survival of monoblastoid cells undergoing TNF‐α‐inuced apoptosis. FASEB J. 13, 793–803 (1999)

Fluorine ion-implanted polystyrene improves growth and viability of vascular smooth muscle cells in culture.

Vascular smooth muscle cells derived from the rat aorta were cultured on unmodified or F(+) ion-implanted polystyrene (5 x 10(12) or 5 x 10(14) ions/cm(2), energy 150 keV). In 1-day-old cultures, the cells adhered to the modified polystyrene in higher numbers and over larger contact areas. Increased resistance of the cells to trypsin-mediated detachment from the growth support indicated an improved adhesion of cells to the modified polymer at later culture intervals. The cells cultured on ion-modified polymers also were larger and had a higher total protein content. By use of immunocytochemistry, several specific protein species were increased, including the cytoskeletal alpha-actin and vimentin and the plasma membrane-associated vinculin, talin, alpha-v integrins, ICAM-1, and VCAM-1, which account for stronger cell-cell and cell-extracellular matrix adhesion. The lower number of cells found floating in the medium suggests that the spontaneous detachment of cells from the modified polystyrene was lower and that the viability of the adhered cell population was higher. As was shown by the two-parameter flow-cytometric measurements of BrdU incorporation and DNA content, as well as by (3)H-thymidine autoradiography, the cell proliferation on samples modified by the dose of 5 x 10(12) ions/cm(2) was similar to that in controls; and at the dose of 5 x 10(14) ions/cm(2), it tended to be even lower. The cells grown on the polymer implanted with the dose of 5 x 10(12) ions/cm(2) responded to a new artificially created cell-free area in a confluent cell layer by more intense migration whereas at the dose of 5 x 10(14) ions/cm(2), the migration ability of cells was similar to that on the unmodified polymer. The data revealed a higher biocompatibility of ion-implanted polystyrene with vascular smooth muscle cells in culture. There was better adhesion, differentiation, and survival, and there was neither excessive migration nor proliferation.

Lead-induced changes in the stabilization of the mouse sperm chromatin

Long-term exposure of male mice to inorganic lead was previously found to reduce their fertility. In the present study chromatin stability in spermatozoa from such mice was investigated by means of quantitative cytochemical analysis. Sperm head sizes were determined and the capacity for nuclear chromatin decondensation (NCD) was evaluated after exposure to a solution of sodium dodecyl sulfate/dithiothreitol. A decreased uptake of propidium iodide (PI), a DNA intercalating dye, was found in spermatozoa from the vas deferens of the lead-exposed mice. However, after thermal denaturation of the DNA, the spermatozoa showed a higher uptake of PI in comparison to those of the controls. After reductive cleavage of S-S bonds with DTT and staining with a thiol-specific reagent (monobromobimane), significantly fewer reactive disulfide bonds were also observed in the spermatozoa. Furthermore, a significant delay in the capacity for NCD was noted. These findings indicate that exposure to lead increases the stabilization of the sperm chromatin, which in turn probably affects the decondensation of the nucleus, thereby interfering with the fertility of the mice.

Rearrangement of nuclear ribonucleoprotein (RNP)-containing structures during apoptosis and transcriptional arrest

Abstract The aim of this paper is to review the data in the literature concerning ribonucleoprotein components during apoptosis, where a major rearrangement of RNPs takes place. In parallel with chromatin changes, the nucleoplasmic constituents (perichromatin fibrils; perichromatin granules; interchromatin granules and nuclear bodies) as well as the nucleoli aggregate into heterogeneous clusters called HERDS, in the interchromatin space. Later, these RNP‐containing structures are extruded from the nucleus and leave the cell within cytoplasmic blebs. We propose also a role for HERDS as markers of irreversible transcriptional arrest.

Nuclear RNA Is Extruded from Apoptotic Cells

During spontaneous apoptosis of thymocytes there is extrusion of ribonucleoproteins (RNPs) from the cell. The aim of this investigation was to elucidate whether the RNP aggregates in apoptotic cells and bodies still contain RNA in an appreciable amount. We demonstrated by specific cytochemical techniques that the aggregates of nuclear RNPs extruded in the cytoplasm of spontaneously apoptotic thymocytes contain RNA in a sufficient amount to be detected cytochemically. These heterogeneous ectopic RNP-derived structures (HERDS) are formed by perichromatin fibrils, interchromatin granules, perichromatin granules, and nucleolar material. The RNA detected inside these clusters should therefore correspond to both mRNA and snRNA as well as to rRNA. We never observed DNA-contaning aggregates in the cytoplasm of apoptotic thymocytes. The presence of RNA in the HERDS that may be released from apoptotic cells suggests that the decrease in the amount of total RNA during apoptosis may be mostly linked to cellular extrusion rather than to degradation of RNA by RNase activities. Another interesting aspect of these results lies in the hypothesis of apoptosis as a possible cause for the presence of autoantibodies in the serum of patients with systemic autoimmune diseases.

DADLE induces a reversible hibernation-like state in HeLa cells.

Abstract[d-Ala(2)-d-Leu(5)-Enkephalin] (DADLE) can induce hibernation when injected into ground squirrels in summer and is able to increase the survival time of explanted organs such as liver and lung. Since cell metabolism is a target of this peptide, we have treated HeLa cells with DADLE and investigated its possible effect on transcription and proliferation as well as the resumption of metabolic activity after treatment. The labelling for Pol I, Pol II and for splicing factors such as snRNPs and SC-35 decreased after treatment as did the nucleolar labelling for UBF. In treated cells, several spherical nuclear bodies were found to be labelled for hnRNPs. In parallel, the number of proliferating cells decreased after treatment with DADLE. After recovery, there was a gradual resumption of cell function: transcription and splicing factors had a distribution similar to that of controls; proliferation resumed; nuclear bodies, representing storage sites for RNPs, disappeared.

Nuclear ribonucleoprotein-containing structures undergo severe rearrangement during spontaneous thymocyte apoptosis. A morphological study by electron microscopy.

Abstract The rearrangement of nuclear ribonucleoprotein (RNP)-containing structures during spontaneous thymocyte apoptosis has been investigated by electron microscopy. Along with chromatin margination and condensation, RNPs segregate into the central portion of the nucleus, in the form of heterogeneous clusters of granules which contain interchromatin and perichromatin granules, perichromatin fibrils, nucleolar components, and probably coiled bodies. In parallel with progressive chromatin condensation and karyorrhexis, granule clusters are then extruded into the cytoplasm and are finally released at the cell surface as membrane-bound cytoplasmic debris, sometimes in association with apparently undamaged organelles such as centrioles. It is likely that this RNP segregation may correlate with a severe impairment of protein synthesis. A similar phenomenon was observed in elongating spermatids, when transcriptional arrest is induced during the process of reversible silencing of the male genome. It may be hypothesized that segregation into heterogeneous granule clusters could be a common mechanism to remove redundant RNP-containing structures from the cell.

Nuclei of aged myofibres undergo structural and functional changes suggesting impairment in RNA processing.

Advancing adult age is associated with a progressive decrease in skeletal muscle mass, strength and quality known as sarcopenia. The mechanisms underlying age-related skeletal muscle wasting and weakness are manifold and still remain to be fully elucidated. Despite the increasing evidence that the progress of muscle diseases leading to muscle atrophy/dystrophy may be related to defective RNA processing, no data on the morpho-functional features of skeletal muscle nuclei in sarcopenia are available at present. In this view, we have investigated, by combining morphometry and immunocytochemistry at light and electron microscopy, the fine structure of myonuclei as well as the distribution and amount of RNA processing factors in skeletal myofibres of biceps brachii and quadriceps femoris from adult and old rats. Results demonstrate that the myonuclei of aged type II fibres show an increased amount of condensed chromatin and lower amounts of phosphorylated polymerase II and DNA/RNA hybrid molecules, clearly indicating a decrease in pre-mRNA transcription rate compared to adult animals. In addition, myonuclei of aged fibres show decreased amounts of nucleoplasmic splicing factors and an accumulation of cleavage factors, polyadenilated RNA and perichromatin granules, suggesting a reduction in the processing and transport rate of pre-mRNA. During ageing, it seems therefore that in rat myonuclei the entire production chain of mRNA, from synthesis to cytoplasmic export, is less efficient. This failure likely contributes to the reduced responsiveness of muscle cells to anabolic stimuli in the elderly.

Two-color fluorescence detection of Poly (ADP-Ribose) Polymerase-1 (PARP-1) cleavage and DNA strand breaks in etoposide-induced apoptotic cells

During apoptosis, the nuclear enzyme Poly(ADP-Ribose) Polymerase-1 (PARP-1) catalyzes the rapid and transient synthesis of poly(ADP-ribose) from NAD+ and becomes inactive when cleaved by caspases. The regulation of these two opposite roles of PARP-1 is still unknown. We have recently investigated PARP-1 activation/degradation in Hep-2 cells driven to apoptosis by actinomycin D. In the present work, we have extended our analysis to the effect of the DNA damaging agent etoposide, and paid attention to the relationship between PARP-1 cleavage and DNA fragmentation. An original fluorescent procedure was developed to simultaneously identify in situ the p89 proteolytic fragment of PARP-1 (by immunolabeling) and DNA degradation (by the TUNEL assay). The presence of p89 was observed both in cells with advanced signs of apoptosis (where the PARP-1 fragment is extruded from the nucleus into the cytoplasm) and in TUNEL-negative cells, with only incipient signs of chromatin condensation; this evidence indicates that PARP-1 degradation in etoposide-treated apoptotic cells may precede DNA cleavage.

RNA/MBNL1-containing foci in myoblast nuclei from patients affected by myotonic dystrophy type 2: an immunocytochemical study.

Myotonic dystrophy type 2 (DM2) is a dominantly inherited autosomal disease with multi-systemic clinical features and it is caused by expansion of a CCTG tetranucleotide repeat in the first intron of the zinc finger protein 9 (ZNF9) gene in 3q21.The expanded-CCUG-containing transcripts are retained in the cell nucleus and accumulate in the form of focal aggregates which specifically sequester the muscleblind-like 1 (MBNL1) protein, a RNA binding factor involved in the regulation of alternative splicing. The structural organization and composition of the foci are still incompletely known. In this study, the nuclear foci occurring in cultured myoblasts from DM2 patients were characterised at fluorescence and transmission electron microscopy by using a panel of antibodies recognizing transcription and processing factors of pre-mRNAs. MBNL1 proved to co-locate in the nuclear foci with snRNPs and hnRNPs, whereas no co-location was observed with RNA polymerase II, the non-RNP splicing factor SC35, the cleavage factor CStF and the PML protein. At electron microscopy the MBNL1-containing nuclear foci appeared as roundish domains showing a rather homogeneous structure and proved to contain snRNPs and hnRNPs. The sequestration of splicing factors involved in early phases of pre-mRNA processing supports the hypothesis of a general alteration in the maturation of several mRNAs, which could lead to the multiple pathological dysfunctions observed in dystrophic patients.