Aging of the cells: Insight into cellular senescence and detection Methods

Cellular theory of aging states that human aging is the result of cellular aging, in which an increasing proportion of cells reach senescence. Senescence, from the Latin word senex, means “growing old,” is an irreversible growth arrest which occurs in response to damaging stimuli, such as DNA damage, telomere shortening, telomere dysfunction and oncogenic stress leading to suppression of potentially dysfunctional, transformed, or aged cells. Cellular senescence is characterized by irreversible cell cycle arrest, flattened and enlarged morphology, resistance to apoptosis, alteration in gene expression and chromatin structure, expression of senescence associated- β-galactosidase (SA-β-gal) and acquisition of senescence associated secretory phenotype (SASP). In this review paper, different types of cellular senescence including replicative senescence (RS) which occurs due to telomere shortening and stress induced premature senescence (SIPS) which occurs in response to different types of stress in cells, are discussed. Biomarkers of cellular senescence and senescent assays including BrdU incorporation assay, senescence associated- β-galactosidase (SA-β-gal) and senescence-associated heterochromatin foci assays to detect senescent cells are also addressed.     

Epidermal tissue homeostasis: Apoptosis and cell emigration as mechanisms of controlled cell deletion in the epidermis,of the toad,Bufo bufo

Summary In normal, non-expanding toad epidermis more cells are produced than needed to replace cells lost by moulting. By implication, cell deletion additional to moulting must take place. This paper deals with the mechanisms by which the surplus of cells is deleted, taking advantage of the fact that the ratio between cell birth rate (K _b) and the rate of desquamation (K _d), which in normal toads is 2 to 3, can be manipulated. In toads deprived of the pars distalis of the pituitary gland it is decreased to 0.2 to 0.3, and in toads with hydrocortisone pellets implanted into the subcutaneous lymph space it is increased to 7 to 10. Thus, structures candidates for the morphological manifestation of the deletion process should occur rarely in toads in which the pars distalis has been removed and frequently in toads with hydrocortisone pellets implanted. Categorization and enumeration of such structures by light microscopy in the epidermis from operated, normal, and hormone-treated toads were performed. The incidence of structures referred to as dark cells and omega-figures were found to correlate relatively well with the K _b/K_d-ratio. A subsequent ultrastructural analysis — on a cell-by-cell basis — of dark cells showed these to reflect various stages of apoptosis. The duration of the apoptotic process was calculated to be approximately 7 h. Light- and electron microscopy of omega-figures combined with histochemical observations of PSA-lectin binding were interpreted as reflecting a release of cells from the basal epidermis and their final elimination within the dermis. It is concluded (i) that apoptosis is an important mechanism of controlled cell deletion, (ii) that emigration to, and elimination in, the dermis is a possible deletion mechanism, and (iii) that necrosis is unlikely to play a role in controlled cell deletion.Supported in part by the Danish National Science Research Council (grant no. 11-6498) (PB)Part of this work was presented at the XVth Meeting of the European Study Group for Cell Proliferation, Sundvollen, Norway, 16–20 September 1987     

Apoptosis-inducing factor of a cytotoxic T cell line: involvement of a secretory phospholipase A2

Natural killer (NK) cells and cytotoxic T lymphocytes (CTLs) kill target cells by the granule-exocytosis pathway and by the engagement of molecules belonging to the tumor necrosis factor family. The involvement of secretory phospholipase A₂ (sPLA₂) in the cytotoxic process has been proposed in NK cells. However, its molecular identity and intracellular localization remain unknown, and its mechanism of action is poorly understood. Here, we have readdressed this issue by studying the cytotoxic activity of whole cell extracts of a CTL line. We observed that inactivation of the perforin-granzyme pathway at 37°C in the presence of 1 mM Ca²⁺ enhanced the ability of CTL extracts to induce apoptosis. This potentiation of cell death was Ca²⁺-dependent, thermo-resistant, and inhibited by 4-bromophenacyl bromide and scalaradial (two inhibitors of sPLA₂). The involvement of an sPLA₂ was confirmed by blocking the pro-apoptotic activity of the Ca²⁺-treated cell extract with an anti-sPLA₂ polyclonal antibody. By cell fractionation assays, we showed that the pro-apoptotic sPLA₂ was localized in the cytoplasmic fraction but not in perforin-rich granules or plasma membrane fractions. Western blotting analysis revealed the presence of four distinct bands of 56, 29.5, 21, and 15 kDa. The highest molecular weight band was consistent with the expression of a group III sPLA2. Taken together, these data indicate that an apoptosis-inducing sPLA₂ is expressed in the cytosol of a CTL cell line and suggest that it plays an effector role in CTL-mediated cytotoxicity. This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), and Programa de Núcleos de Excelência (PRONEX–CNPq).     

Role of HRTPT in kidney proximal epithelial cell regeneration: Integrative differential expression and pathway analyses using microarray and scRNA-seq

Damage to proximal tubules due to exposure to toxicants can lead to conditions such as acute kidney injury (AKI), chronic kidney disease (CKD) and ultimately end-stage renal failure (ESRF). Studies have shown that kidney proximal epithelial cells can regenerate particularly after acute injury. In the previous study, we utilized an immortalized in vitro model of human renal proximal tubule epithelial cells, RPTEC/TERT1, to isolate HRTPT cell line that co-expresses stem cell markers CD133 and CD24, and HREC24T cell line that expresses only CD24. HRTPT cells showed most of the key characteristics of stem/progenitor cells; however, HREC24T cells did not show any of these characteristics. The goal of this study was to further characterize and understand the global gene expression differences, upregulated pathways and gene interaction using scRNA-seq in HRTPT cells. Affymetrix microarray analysis identified common gene sets and pathways specific to HRTPT and HREC24T cells analysed using DAVID, Reactome and Ingenuity software. Gene sets of HRTPT cells, in comparison with publicly available data set for CD133+ infant kidney, urine-derived renal progenitor cells and human kidney-derived epithelial proximal tubule cells showed substantial similarity in organization and interactions of the apical membrane. Single-cell analysis of HRTPT cells identified unique gene clusters associated with CD133 and the 92 common gene sets from three data sets. In conclusion, the gene expression analysis identified a unique gene set for HRTPT cells and narrowed the co-expressed gene set compared with other human renal–derived cell lines expressing CD133, which may provide deeper understanding in their role as progenitor/stem cells that participate in renal repair.     

From oocyte maturation to the in vitro cell cycle: the history of discoveries of Maturation-Promoting Factor (MPF) and Cytostatic Factor (CSF)

This article briefly reviews the classical cell cycle studies using oocytes and zygotes of mainly amphibians in the past century. The discussions are focused on the investigations into the cytoplasmic factors that regulate meiosis during oocyte maturation and the initiation of mitosis during fertilisation, which were carried out in the author's lab between 1967 and 1987. This chronicle traces the development of the problems and the direction in which their solutions were attempted in the course of these investigations. The author tries to answer the following questions: why he decided to study oocyte maturation, how he discovered progesterone as a maturation-inducing hormone, how he discovered and characterised the cytoplasmic regulators of the cell cycle, Maturation-Promoting Factor (MPF) and Cyto-Static Factor (CSF), and how he invented the method of observing cell cycle processes in a cytoplasmic extract in vitro.     

Biomarkers for early detection of high risk cancers: From gliomas to nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NpC) is a malignant disease associated with Epstein-Barr virus infection, and often diagnosed at an advanced stage. This significantly curtails patient survival. We hypothesize that a panel of biomarkers can be assembled to assess NpC incidence, early detection, and tumor progression during therapeutic intervention. Our thesis rests on a model of successfully predicting high-risk gliomas by means of a carefully crafted panel of molecular mitotic biomarkers (i.e., securin, survivin and MCM2). The strategy we propose holds strong promise for prevention and cure of NpC. The approach we propose seeks to identify certain biomarkers from viral materials, patient tissues and assessment of related diseases, whose signatures, taken together, will be endowed with some degree of congruency, or sense of a coordinated language (i.e., “votes”). Biomarker “voting” will then permit to outline a broad coordinated molecular map for the molecular and epigenetic characterization of each individual patient''s NpC tumor. We will draw on the process of contrasting biomarkers in health and disease, which rests on the auto-proteomic concept particularly relevant in high-risk cancer individuals, such as is the case for NpC. In brief we defend, current advances in human proteome profiling proffers the possibility of having individual baseline proteomic profiles using local body fluids (e.g., saliva, nasal secretions, sputum) or systemic fluids (e.g., plasma, serum, cerebrospinal fluid) to unravel a personalized molecular map for high-risk NpC individuals. Regular check-up will monitor for new or impending manifestations of NpC, and provide a secure assessment of incidence and early detection.     

Apoptosis shifts to necrosis via intermediate types of cell death by a mechanism depending on c-myc and bcl-2 expression

Hypoxic and chemical hypoxia (antimycin A) commits cultured rat fibroblasts (Rat-1) towards apoptosis, necrosis or an intermediate form of cell death (aponecrosis) depending on the degree of hypoxia. Aponecrosis also occurs in vivo. Here, we demonstrate that c-myc and bcl-2, two proto-oncogenes known to lower or to enhance, respectively, the apoptotic threshold, also affect the type of cell death: apoptosis shifts to aponecrosis and aponecrosis to necrosis, depending on c-myc or bcl-2 expression and the antimycin A concentration (100–400 M). In cells with basal gene expression, apoptosis shifts to aponecrosis/necrosis at 300 M antimycin A (middle hypoxia). Overexpression of c-myc markedly increases cumulative cell death in response to antimycin A and lowers the antimycin A concentration required to shift apoptosis to aponecrosis/necrosis from 300 M to 100 M (low hypoxia). Overexpression of bcl-2 elicits the opposite effect, decreasing cumulative cell death in response to antimycin A and raising the drug concentration required to shift apoptosis to aponecrosis/necrosis to 400 M (high hypoxia). The passage from one to the other form of cell death involves various aponecrotic features with observed intermediate aspects between apoptosis and necrosis, a progressive increase in necrotic features being correlated with an increase in antimycin A concentration. The mechanism underlying the various effects of c-myc and bcl-2 on cell-death type has been related to the ability of these genes to counteract, to various extents, the ATP decrease occurring in response to different degrees of chemical hypoxia.The first two authors contributed equally to this workThis work was supported by grants from the Associazione Italiana per la Ricerca sul Cancro (AIRC, Milano), CNR/MIUR (Grant Progetto Finalizzato Oncologia), Ente Cassa di Risparmio di Firenze and Ministero dellIstruzione, dellUniversità e della Ricerca (MIUR, Cofin 2003). Andrea Lapucci is supported by a fellowship from the Federazione Italiana per la Ricerca sul Cancro (FIRC, Milano)